Compositions with permeation enhancers for drug delivery

ABSTRACT

The present invention provides compositions and methods for delivery of therapeutic agents across an barrier. The compositions include a therapeutic agent (e.g., antimicrobial agent, antibiotic, or anesthetic agent), a permeation enhancer which increases the flux of the therapeutic agent across the barrier, and a matrix forming agent. The matrix forming agent forms a gel at a suitable gelation temperature and rheological properties for use in drug delivery, and in some cases, the gelation temperature and rheological properties are not significantly changed from those of the composition without the permeation enhancer. The invention also provides a matrix forming agent and compositions thereof. Such compositions are particularly useful in the treatment of otitis media. Methods of treatment, methods of delivery, and kits for the compositions described herein are also provided.

RELATED APPLICATIONS

This application is a continuation of U.S. patent application U.S. Ser.No. 15/749,951, filed Feb. 2, 2018, which is a national stage filingunder 35 U.S.C. 371 of International Patent Application Serial No.PCT/US2016/045908, filed Aug. 5, 2016, entitled “COMPOSITIONS WITHPERMEATION ENHANCERS FOR DRUG DELIVERY”, which claims the benefit of thefiling under 35 U.S.C. 119(e) to of U.S. Provisional Application Ser.No. 62/201,199, filed Aug. 5, 2015, the entire contents of which are isincorporated herein by reference. International ApplicationPCT/US2016/045908 was published under PCT Article 21(2) in English.

FEDERALLY SPONSORED RESEARCH

This invention was made with government support under Grant Nos.GM073626 and DC009986 awarded by the National Institutes of Health. Thegovernment has certain rights in the invention.

BACKGROUND

Twelve to 16 million physician visits per year in the United States areattributed to otitis media (OM), making it the most common specificallytreated childhood disease. [1] Acute OM (AOM) has a prevalence of 90%within the first 5 years of life, [2] and 90-95% of all U.S. childrenhave at least one documented middle ear effusion by age 2. [3] 25%percent of all prescriptions written for children are for treatment ofacute otitis media. Recurrence of the disease is also striking, with onethird of all children in the U.S. having 6 or more episodes of AOM byage 7. [4] Moreover, epidemiological studies suggest that the prevalenceof recurrent OM among children, particularly infants, is on the rise.[5] The incidence of OM in children of other industrialized nations issimilar to that in the U.S. In the developing world, OM remains asignificant cause of childhood mortality due to the development ofchronic suppurative otitis media which frequently results in permanenthearing sequelae, and due to intracranial complications estimated toresult in more than 25,000 deaths worldwide. [6]

Acute OM is the most common reason for antimicrobial prescribing in U.S.children and due to the high prevalence of disease and frequentrecurrences is believed to be partially responsible for the ongoingincrease in antibiotic resistance among pathogenic bacteria. Despite thesuccess in reducing antimicrobial use in children by approximately 25%over the past decade, the increase in antimicrobial resistance hascontinued.

Present treatment of ear infections consists of systemic oralantibiotics, a treatment which requires multiple doses over 5-10 daysand systemic exposure to antibiotics. The rise in antibiotic resistance,coupled with the many multifactorial etiology of OM pose difficulties indiagnosis and treatment of OM. Furthermore, current treatment presents anumber of drawbacks including patient compliance issues due togastrointestinal side effects, lack of an effective concentration ofdrug at the site of infection, and the potential for opportunisticinfections. Even after acute signs of infection subside, generallywithin 72 hours, the root cause of the infection may persist for theremainder of the treatment, and beyond, even up to 2 months. Thus,making compliance with a physician's prescription important to preventreoccurrence of infection.

Local, sustained delivery of active therapeutics directly to the middleear for the treatment of OM could allow for much higher concentrationsof the drug in the middle ear than from systemic administration, whileminimizing systemic exposure and its adverse effects. However, thetympanic membrane (TM), while only 10 cell-layers thick, presents abarrier that is largely impermeable to all but the smallest, moderatelyhydrophobic molecules. Despite being the thinnest layer of skin, it isstill a barrier to trans-tympanic membrane diffusion. Therefore, thedirect treatment of middle ear infections is problematic. Theshortcomings of the current treatment of ear diseases, such as middleear infections, suggest the need for a new treatment which isnoninvasive and direct acting.

SUMMARY

Provided herein are compositions and methods aimed at non-invasivetrans-tympanic otitis media (OM) treatment with sustained drug fluxacross the tympanic membrane (TM) (See, e.g., FIG. 1). Chemicalpermeation enhancers (CPEs), commonly employed for trans-dermaldelivery, can enable such a trans-tympanic flux. In certain embodiments,a single application of an optimized formulation could provide highconcentrations of antibiotics localized to the middle ear, resulting ineradication of bacterial otitis media without the drawbacks of oraltherapy. Such formulations may also useful in the treatment of otherdiseases of the ear requiring drug delivery across the tympanicmembrane.

Typical OM treatments consists of a 10-day course of broad spectrum oralantibiotics. The widespread use of systemic antibiotics against adisease of such high prevalence and recurrence is believed to bepartially responsible for the ongoing increase in antibiotic resistanceseen in pathogenic bacteria in the nasopharynx. In most cases,antibiotic-resistant infections like pneumonia, skin, soft tissue, andgastrointestinal infections require prolonged and/or costliertreatments, extend hospital stays, necessitate additional doctor visitsand healthcare use, and result in greater disability and death comparedwith infections that are easily treatable with antibiotics. Compliancewith multi-dose regimens can also be difficult in some parts of theworld. Compliance and antibiotic resistance may also be more problematicin the long-term prophylaxis of recurrent OM. An effective sustainedlocal therapy could address the issue of compliance, affect thedevelopment of drug-resistant and chronic suppurative otitis media, andreduce the need for tympanostomy tube placement (devices implanted inthe TM to enhance middle ear drainage in recurrent OM). [8]

The TM is a tri-layer membrane whose outer layer is a stratifiedsquamous keratinizing epithelium continuous with the skin of theexternal auditory canal. The inner-most layer is a simple cuboidalmucosal epithelium. Between these epithelia is a layer of fibro-elasticconnective tissue and associated blood vessels and nerves. The human TMis only about 100 μm thick, but the 6-10 cell layer outer epitheliumforms an impenetrable barrier against all but the smallest lipophilicmolecules due to its keratin- and lipid-rich stratum corneum. [11]

Localized, sustained drug delivery directly to target tissues hasseveral advantages over systemic application, including fewer adversesystemic effects, smaller quantities of drug used, potentially bettertherapeutic outcomes, and reduced costs. The impermeability of the TM isa central challenge for the development of local therapies.

Chemical permeation enhancers (CPEs) are used to safely increase smallmolecule flux in transdermal drug delivery. Several are FDA approved foruse in humans. These agents are often surfactants, comprising aheterogeneous group of amphiphilic organic molecules with hydrophilicheads and hydrophobic tails. Several classes of surfactants have beenstudied. Surfactants reversibly modify lipids by adsorption atinterfaces and removal of water-soluble agents that act as plasticizers.Cationic surfactants are known to produce greater increases in permeantflux than anionic surfactants, which in turn increase permeability morethan nonionic surfactants. A broad range of non-surfactant chemicalenhancers (e.g., terpenes) has also been used with mechanisms of actionincluding denaturation of proteins within and between keratinocytes,and/or modification or disruption of lipids that results in increasedlipid bilayer fluidity.

In a composition provided herein, the therapeutic agents and permeationenhancers are combined with matrix forming agents, to form compositionswhich form a hydrogels under suitable conditions. Such conditions mayinclude exposure to body heat during administration (e.g., in the earcanal), or following mixing of two components of the composition ormatrix-forming agent. The matrix forming agent is a compound or mixtureof compounds that forms a gel after administration. The compositions aregenerally liquid at ambient conditions, however, once administered to asubject, the matrix forming agent or combination of matrix formingagents causes a phase transition to a hydrogel. Hydrogels have a highlyporous structure that allows for the loading of drugs and other smallmolecules, and subsequent drug elution out of the gel creates a highlocal concentration in the surrounded tissues over an extended period.In certain embodiments, the drugs are loaded in the liquid composition.Hydrogels can conform and adhere to the shape of the surface to whichthey are applied and tend to be biocompatible.

For the compositions provided herein, the combination of the permeationenhancer with the matrix forming agent and therapeutic agent provides acomposition with improved flux of the therapeutic agent, and alsoimproved, or not significantly impaired, properties of the resultinghydrogel relative to the hydrogel formed by the composition in theabsence of the permeation enhancer. For example, the phase transitiontemperature of the composition with the permeation enhancer may be lowerthan the composition without the permeation enhancer, or even if higher,may still fall into a useful range for formation of a hydrogel uponexposure to a biological surface (e.g., a phase transition temperaturebetween about 0° C. and about 37° C. As another example, the storagemodulus and/or loss modulus of the composition with the permeationenhancer may be about the same (e.g., within about 20%) as for thecomposition without the permeation enhancer, or the storage modulus ofthe composition with the permeation enhancer may be higher than thecomposition without the permeation enhancer. As another example, thestorage modulus and/or loss modulus of the composition with thepermeation enhancer may be about the same (e.g., within about 20% or 3kPa, whichever is greater) as for the composition without the permeationenhancer, or the storage modulus of the composition with the permeationenhancer may be higher than the composition without the permeationenhancer. For the compositions provided herein, the combination of thepermeation enhancer with the matrix forming agent and therapeutic agentprovides a composition with improved flux of the therapeutic agent, andadditional improved properties including, but not limited to extendeddrug release, adherence of the composition to the tympanic membrane overtime, degradation, or combinations thereof, and also improved, or notsignificantly impaired, properties of the resulting hydrogel relative tothe hydrogel formed by the composition in the absence of the permeationenhancer.

In one aspect, provided herein are compositions comprising:

-   -   (a) a therapeutic agent or a combination of therapeutic agents;    -   (b) a permeation enhancer or a combination of permeation        enhancers, wherein the permeation enhancer or combination of        permeation enhancers increases the flux of the therapeutic agent        or combination of therapeutic agents across a barrier; and    -   (c) a matrix forming agent or a combination of matrix forming        agents, wherein the matrix forming agent or combination of        matrix forming agents comprises a polymer;        wherein:

the composition forms a gel at temperatures above a phase transitiontemperature; and

the phase transition temperature is less than about 37° C.;

and at least one of conditions (i), (ii), and (iii) are met:

-   -   (i) the phase transition temperature of the composition is less        than the phase transition temperature of a reference composition        plus about 5° C.;    -   (ii) the storage modulus of the composition is greater than        about 15% of the storage modulus of the reference composition at        a temperature of about 37° C.; and    -   (iii) the loss modulus of the composition is between about 80%        and about 120% of the loss modulus of the reference composition        at a temperature of about 37° C.;        wherein the reference composition is the composition in the        absence of (b) the permeation enhancer or combination of        permeation enhancers.

In one aspect, provided herein are compositions comprising:

-   -   (a) a therapeutic agent or a combination of therapeutic agents;    -   (b) a permeation enhancer or a combination of permeation        enhancers, wherein the permeation enhancer or combination of        permeation enhancers increases the flux of the therapeutic agent        or combination of therapeutic agents across a barrier; and    -   (c) a matrix forming agent or a combination of matrix forming        agents, wherein the matrix forming agent or combination of        matrix forming agents comprises a polymer;        wherein:

the composition forms a gel at temperatures above a phase transitiontemperature; and

the phase transition temperature is less than about 37° C.;

and at least one of conditions (i), (ii), and (iii) are met:

-   -   (i) the phase transition temperature of the composition is less        than the phase transition temperature of a reference composition        plus about 5° C.;    -   (ii) the storage modulus of the composition is greater than        about 15% of the storage modulus of the reference composition or        greater than about 500 Pa, whichever is smaller, at a        temperature of about 37° C.; and    -   (iii) the loss modulus of the composition is between about 15%        and about 150% of the loss modulus of the reference composition        at a temperature of about 37° C.;        wherein the reference composition is the composition in the        absence of (b) the permeation enhancer or combination of        permeation enhancers.

In certain embodiments, condition (i), the phase transition temperatureof the composition is less than the phase transition temperature of thereference composition plus about 5° C., is met. In certain embodiments,condition (ii), the storage modulus of the composition is greater thanabout 15% of the storage modulus of the reference composition, is met.In certain embodiments, condition (ii), the storage modulus of thecomposition is greater than about 15% of the storage modulus of thereference composition, or greater than about 500 Pa, whichever issmaller, is met. In certain embodiments, condition (ii), the storagemodulus of the composition is greater than about 15% of the storagemodulus of the reference composition, or greater than about 1000 Pa,whichever is smaller, is met. In certain embodiments, condition (iii),the loss modulus of the composition is between about 80% and about 120%of the loss modulus of the reference composition, is met. In certainembodiments, condition (iii), the loss modulus of the composition isbetween about 15% and about 150% of the loss modulus of the referencecomposition, is met. In certain embodiments, both conditions (i) and(ii) are met. In certain embodiments, both conditions (ii) and (iii) aremet. In certain embodiments, both conditions (i) and (iii) are met. Incertain embodiments, each of conditions (i), (ii), and (iii) are met.

In certain embodiments, the polymer is biodegradable. In certainembodiments, the polymer is a copolymer. In certain embodiments, thecopolymer is biodegradable or comprises biodegradable monomers. Incertain embodiments, the copolymer is a block copolymer. In certainembodiments the copolymer comprises at least one block of hydrophobicmonomers. In certain embodiments, the copolymer comprises at least oneblock of hydrophobic monomers, and at least one block of non-hydrophobicmonomers.

In certain embodiments, the copolymer comprises a vinylic polymer (e.g.PE, PVC, PVDC, PS), a polyacrylate (e.g., polyacrylic acidpolymethacrylic acid), a polyether (e.g., PEO, PPO, POM), afluoropolymer (e.g., PTFE), a polysiloxane (e.g., PDMS), apolysaccharide (e.g., cellulose, dextran, hyaluronic acid, chitosan), apolyester (e.g, PET, a polyhydroxyalkanoate (e.g., PHB)), a polyamide(e.g., poly(lactic acid), poly(glycolic acid)), a polyphosphoester, apolyurethane, or a polycarbonate, or copolymers of combinations thereof.In certain embodiments, the copolymer comprises polyethylene oxide,polypropylene oxide, a poloxamer, poloxamer 407, poloxamer 188, apoloxamine, methylcellulose, hydroxypropyl methylcellulose,ethyl(hydroxyethyl) cellulose, xyloglucan, cellulose, acetate phthalate,latex, poly(acrylic acid), N-isopropylacrylamide-based systems,hyaluronic acid, chitosan, dextran, or gellan gum, or a derivativethereof, or a copolymer of a combination thereof. In certainembodiments, the copolymer comprises a poloxamer. In some embodiments,the copolymer comprises poloxamer 407. In certain embodiments, thecopolymer comprises phosphoester monomers. In certain embodiments, thecopolymer comprises a poloxamer and phosphoester monomers.

In certain embodiments, the composition has a high degree ofhydrophobicity. In certain embodiments, the composition is opticallytransparent.

In certain embodiments, the phase transition temperature of thecomposition is at or below the body temperature of a subject. In certainembodiments, the phase transition temperature of the composition isbetween about 10° C. and about 40° C. In certain embodiments, the phasetransition temperature of the composition is between about 20° C. andabout 40° C. In certain embodiments, the phase transition temperature ofthe composition is less than the phase transition temperature of thesame composition without the permeation enhancer plus about 5° C.

In certain embodiments, the composition is useful in treating a disease.In some embodiments, the composition is useful in treating an infectiousdisease. In some embodiments, the composition is useful in treating anear disease (e.g., the barrier is the tympanic membrane). In someembodiments, the composition is useful in treating otitis media.

In another aspect, provided herein are compositions for treating aninfectious disease or ear disease comprising:

-   -   (a) a therapeutic agent or a combination of therapeutic agents;    -   (b) a permeation enhancer or a combination of permeation        enhancers, wherein the permeation enhancer or combination of        permeation enhancers increases the flux of the therapeutic agent        or combination of therapeutic agents across a barrier; and    -   (c) a matrix forming agent or a combination of matrix forming        agents, wherein the matrix forming agent or combination of        matrix forming agents comprises a copolymer comprising        phosphoester monomers.

The therapeutic agent may be an antibiotic agent, anesthetic agent,anti-inflammatory agent, analgesic agent, anti-fibrotic agent,anti-sclerotic agent, or anticoagulant agent. In certain embodiments,the therapeutic agent is an antibiotic selected form the groupconsisting of ciprofloxacin, cefuroxime, cefadroxil, cefazolin,cefalotin, cefalexin, cefaclor, cefamandole, cefoxitin, cefprozil,cefuroxime, cefixime, cefdinir, cefditoren, cefoperazone, cefotaxime,cefpodoxime, ceftazidime, ceftibuten, ceftizoxime, ceftriaxone,cefepime, ceftobiprole, enoxacin, gatifloxacin, levofloxacin,lomefloxacin, moxifloxacin, norfloxacin, ofloxacin, trovafloxacin,bacitracin, colistin, polymyxin B, azithromycin, clarithromycin,dirithromycin, erythromycin, roxithromycin, troleandomycin,telithromycin, spectinomycin, amoxicillin, ampicillin, azlocillin,carbenicillin, cloxacillin, dicloxacillin, flucloxacillin, mezlocillin,meticillin, nafcillin, oxacillin, penicillin, piperacillin, ticarcillin,mafenide, sulfacetamide, sulfamethizole, sulfasalazine, sulfisoxazole,trimethoprim, and trimethoprim-sulfamethoxazole. In some embodiments,the antibiotic is ciprofloxacin. In some embodiments, the antibiotic isamoxicillin, azithromycin, cefuroxime, ceftriaxone, or trimethoprim. Insome embodiments, the antibiotic is levofloxacin.

The permeation enhancer may be a surfactant, terpene, amino amide, aminoester, azide-containing compound, alcohol, or anesthetic agent. Thepermeation enhancer may be a surfactant, terpene, amino amide, aminoester, azide-containing compound, alcohol, pyrrolidone, sulfoxide, fattyacid, or anesthetic agent. In some embodiments, the permeation enhanceris a surfactant (e.g., cationic surfactant, anionic surfactant, nonionicsurfactant). In some embodiments, the permeation enhancer is a terpene.In some embodiments, the composition comprises a surfactant permeationenhancer and a terpene permeation enhancer.

In certain embodiments, the permeation enhancer is sodium dodecylsulfate, ammonium lauryl sulfate, sodium lauryl sulfate, cetyltrimethylammonium bromide, cetylpyridinium chloride, benzethoniumchloride, cocamidopropyl betaine, cetyl alcohol, oleyl alcohol, octylglucoside, decyl maltoside, sodium octyl sulfate, sodium decyl sulfate,sodium tetradecyl sulfate, sodium heptadecyl sulfate, sodium eicosylsulfate, nicotine sulfate, sodium taurocholic sulfate, dimethylsulfoxide, sodium tridecyl phosphate; decyldimethyl ammonio propanesulfonate, chembetaine oleyl, myristyldimethyl ammonio propanesulfonate; benzyl pyridinium chloride, dodecyl pyridinium chloride,cetyl pyridinium chloride, benzyldimethyl dodecyl ammonium chloride,benzyldimethyl dodecyl ammonium chloride, benzyldimethyl myristylammonium chloride, benzyldimethyl stearyl ammonium chloride,octyltrimethylammonium bromide, dodecyltrimethylammonium bromide,Polysorbate 20, Polysorbate 40, Polysorbate 60, or Polysorbate 80. Incertain embodiments, the permeation enhancer is sodium octyl sulfate,sodium dodecyl sulfate, octyl trimethylammonium bromide, dodecyltrimethylammonium bromide, Polysorbate 20, or Polysorbate 80. In someembodiments, the permeation enhancer is sodium dodecyl sulfate.

In certain embodiments, the permeation enhancer is sodium lauroylsarcosinate, sorbitan monooleate, octoxynol-9, diethyl sebacate, sodiumpolyacrylate (2500000 molecular weight (MW)), or octyldodecanol.

In certain embodiments, the permeation enhancer is an azone-likecompound. In certain embodiments, the permeation enhancer is a compoundsimilar to azone (e.g., laurocapram) of the formula:

In certain embodiments, the permeation enhancer is a compound containingpiperazine. In certain embodiments, the permeation enhancer is1-benzyl-4-(2-((1,1-biphenyl)-4-yloxy)ethyl)piperazine.

In certain embodiments, the permeation enhancer is a terpene (e.g.,limonene). In certain embodiments, the permeation enhancer is limonene,cymene, pinene, camphor, menthol, comphone, phellandrine, sabinene,terpinene, borneol, cineole, geraniol, linalol, pipertone, terpineol,eugenol, eugenol acetate, safrole, benzyl benzoate, humulene,beta-caryophylene, eucakytol, hexanoic acid, octanoic acid, decanoicacid, undecanoic acid, dodecanoic acid, tridecanoic acid, myristic acid,palmitic acid, stearic acid, oleic acid, linoleic acid, linolenic acid,cholic acid; ethyl undecanoate, methyl laurate, methyl myristate,isopropyl myristate, isopropyl palmitate, palmityl palmitate, diethylsebaccate, glyceryl monolaurate, glyceryl monooleate, or ethylpiperazinecarboxylate. In some embodiments, the permeation enhancer is limonene.

In certain embodiments, the permeation enhancer is bupivacaine,tetracaine, procaine, proparacaine, propoxycaine, dimethocaine,cyclomethycaine, chloroprocaine, benzocaine, lidocaine, prilocaine,levobupivicaine, ropivacaine, dibucaine, articaine, carticaine,etidocaine, mepivacaine, piperocaine, or trimecaine. In someembodiments, the permeation enhancer is bupivacaine.

In some embodiments, the permeation enhancer is a combination of asurfactant and a terpene. In some embodiments, the permeation enhanceris a combination of a surfactant, a terpene, and an anesthetic. In someembodiments, the permeation enhancer is a combination of a surfactantselected from: sodium octyl sulfate, sodium dodecyl sulfate, octyltrimethylammonium bromide, dodecyl trimethylammonium bromide,Polysorbate 20, and Polysorbate 80; and a terpene. In some embodiments,the permeation enhancer is a combination of a surfactant selected from:sodium octyl sulfate, sodium dodecyl sulfate, octyl trimethylammoniumbromide, dodecyl trimethylammonium bromide, Polysorbate 20, andPolysorbate 80; and limonene. In some embodiments, the permeationenhancer is sodium dodecyl sulfate, limonene, or bupivacaine, or acombination thereof. In some embodiments, the permeation enhancer is acombination of sodium dodecyl sulfate and limonene. In some embodiments,the permeation enhancer is a combination of sodium dodecyl sulfate,limonene, and bupivacaine.

The compositions may also include additional therapeutic agents,including anti-inflammatory agents (e.g., dexamethasone), anesthetics(e.g., bupivacaine), or β-lactamase inhibitors. In some embodiments, atherapeutic agent or additional therapeutic agent also acts as apermeation enhancer. In some embodiments, an amino amide (e.g.,bupivacaine) or amino ester (e.g., tetracaine) local anesthetic acts asboth a permeation enhancer and a therapeutic agent. In some embodiments,the composition comprises an amino amide (e.g., bupivacaine) or aminoester (e.g., tetracaine) local anesthetic acting as both a permeationenhancer and a therapeutic agent, and does not comprise an additionaltherapeutic agent. In some embodiments, the composition comprisesbupivacaine acting as both a permeation enhancer and a therapeuticagent, and does not comprise an additional therapeutic agent.

In certain embodiments, the therapeutic agents comprises between about0.01 percent to about 10 percent of the composition. In certainembodiments, the percent weight of permeation enhancer in thecomposition is between about 0.1% to about 1%, between about 1% to about3%, or between about 3% to about 10%. In certain embodiments, thepercent weight of matrix forming agent in the composition is betweenabout 1% to about 10%, between about 10% to about 20%, between about 20%to about 30%, between about 30% to about 40%, or between about 40% toabout 50%. Unless otherwise state, percent compositions herein refer toweight of the component per volume of the composition.

In certain embodiments, the matrix forming agent comprises monomers ofFormula (M):

In certain embodiments, the matrix forming agent is a block copolymer ofFormula (I):

For Formulae (M) and (I):

-   -   each occurrence of Y is independently —R¹ or -L²R²;    -   each occurrence of R¹ is independently hydrogen, optionally        substituted alkyl, optionally substituted alkenyl, optionally        substituted alkynyl, optionally substituted aryl, or optionally        substituted heteroaryl;    -   each occurrence of L² is independently a bond, optionally        substituted alkylene, optionally substituted alkenylene,        optionally substituted alkynylene, optionally substituted        heteroalkylene, optionally substituted heteroalkenylene, or        optionally substituted heteroalkynylene;    -   each occurrence of R² is independently optionally substituted        acyl, optionally substituted carbocyclyl, optionally substituted        heterocyclyl, optionally substituted aryl, optionally        substituted heteroaryl, —OR^(b), —N(R^(b))₂, or an oxygen        protecting group;    -   each occurrence of R³ is independently optionally substituted        alkyl, optionally substituted alkenyl, optionally substituted        alkynyl, optionally substituted aryl, optionally substituted        heteryaryl, optionally substituted acyl, —OR^(b), or —N(R^(b))₂;    -   each occurrence of R^(b) is independently optionally substituted        alkyl, optionally substituted alkenyl, optionally substituted        alkynyl, optionally substituted carbocyclyl, optionally        substituted heterocyclyl, optionally substituted aryl,        optionally substituted heteroaryl, optionally substituted acyl,        an oxygen protecting group, or a nitrogen protecting group, or        two R^(b) taken together with the nitrogen to which they are        attached form an optionally substituted heterocyclic or        optionally substituted heteroaryl ring;    -   each of G¹ and G² is independently hydrogen, optionally        substituted alkyl, optionally substituted aryl, or optionally        substituted heteroaryl, optionally substituted acyl, optionally        substituted phosphate, or an oxygen protecting group; and    -   each of p, q, r, s, and t is independently an integer between 0        and 200, inclusive, wherein the sum of p and t is at least 1,        and the sum of q, r, and s is at least 1.

In certain embodiments, the matrix forming agent comprises a blockcopolymer comprising poloxamer 407 (P407), or a similar analog, linkedby its terminal hydroxyl groups to polyphosphoester (PPE) blocks (SeeFIG. 7). Hydrophobic PPE side chains may increase the hydrogel modulus,decrease erosion of the hydrogel in situ, enhance micelle packing duringgelation, decrease the gelation temperature, or a combination thereof.The thermo-responsiveness and bioadhesion characteristics of thecomposition may be tuned by selection of side chains (e.g., alkyl sidechains, acryl side chains).

In another aspect, provided herein are a matrix forming agent or acombination of matrix forming agents of Formula (I′):

wherein each occurrence of Y is independently —R¹ or -L²R²;

-   -   each occurrence of R¹ is independently hydrogen, optionally        substituted alkyl, optionally substituted alkenyl, optionally        substituted alkynyl, optionally substituted aryl, or optionally        substituted heteroaryl;    -   each occurrence of L² is independently a bond, optionally        substituted alkylene, optionally substituted alkenylene,        optionally substituted alkynylene, optionally substituted        heteroalkylene, optionally substituted heteroalkenylene, or        optionally substituted heteroalkynylene;    -   each occurrence of R² is independently optionally substituted        acyl, optionally substituted carbocyclyl, optionally substituted        heterocyclyl, optionally substituted aryl, optionally        substituted heteroaryl, —OR^(b), —N(R^(b))₂, or an oxygen        protecting group;    -   each occurrence of R^(3A) is independently hydrogen, optionally        substituted alkyl, optionally substituted alkenyl, optionally        substituted alkynyl, optionally substituted aryl, optionally        substituted heteryaryl, optionally substituted acyl, —OR^(b), or        —N(R^(b))₂;    -   each occurrence of R^(b) is independently optionally substituted        alkyl, optionally substituted alkenyl, optionally substituted        alkynyl, optionally substituted carbocyclyl, optionally        substituted heterocyclyl, optionally substituted aryl,        optionally substituted heteroaryl, optionally substituted acyl,        an oxygen protecting group, or a nitrogen protecting group, or        two R^(b) taken together with the nitrogen to which they are        attached form an optionally substituted heterocyclic or        optionally substituted heteroaryl ring;    -   each of G^(1A) and G^(2A) is independently hydrogen, halogen,        optionally substituted amine, optionally substituted alkyl,        optionally substituted aryl, or optionally substituted        heteroaryl, optionally substituted acyl, optionally substituted        phosphate, or an oxygen protecting group; and

each of p, q, r, s, and t is independently an integer between 0 and 200,inclusive, wherein the sum of p and t is at least 1, and the sum of q,r, and s is at least 1.

In another aspect, provided herein are compositions comprising a matrixforming agent or a combination of matrix forming agents of Formula (I′),

In another aspect, provided herein are methods for treating aninfectious disease comprising administering a composition comprising atherapeutic agent, permeation enhancer, and a matrix forming agent, asdescribed herein, to a subject in need thereof.

In another aspect, provided herein are methods for treating an eardisease comprising administering a composition comprising a therapeuticagent, permeation enhancer, and a matrix forming agent, as describedherein, to a subject in need thereof. In certain embodiments, thecomposition is administered into the ear canal or to the tympanicmembrane. In certain embodiments, the disease is otitis media. Incertain embodiments, the disease is an ear infection. In certainembodiments, the disease is a bacterial infection (e.g., a H.influenzae, S. pneumoniae, or M. catarhallis infection).

In another aspect, provided herein are methods for eradicating a biofilmcomprising administering to a subject in need thereof, or contacting abiofilm with, a composition described herein.

In another aspect, provided herein are methods for inhibiting theformation of a biofilm comprising administering to a subject in needthereof, or contacting a surface with, a composition described herein.

In an additional aspect, provided herein are methods for delivering acomposition described herein, the method comprising administering intoan ear canal of a subject the composition, wherein the compositioncontacts the surface of a tympanic membrane. The composition may beadministered with an eye dropper, syringe, double barrel syringe, orcatheter (e.g., angiocatheter).

In an additional aspect, the provided herein are kits comprising acontainer, a composition described herein, and instructions foradministering the composition to a subject in need thereof. The kit mayfurther comprise a device for administration of the composition to asubject, such as a dropper, syringe, catheter, double barrel syringe, orcombination thereof.

The compositions, composition components (e.g., matrix forming agents,therapeutic agents, and permeation enhancers), methods, kits, and usesof the present disclosure may also incorporate any feature described in:Khoo et al., Biomaterials. (2013) 34, 1281-8; U.S. Pat. No. 8,822,410;U.S. patent application Ser. No. 12/993,358, filed May 19, 2009; U.S.patent application Ser. No. 11/734,537; filed Apr. 12, 2007; WIPO PatentApplication No. PCT/US2009/003084, filed May 19, 2009, and WIPO PatentApplication No. PCT/US2007/009121, filed Apr. 12, 2007, each of which isincorporated herein by reference.

The details of certain embodiments of the invention are set forth in theDetailed Description of Certain Embodiments, as described below. Otherfeatures, objects, and advantages of the invention will be apparent fromthe Definitions, Examples, Figures, and Claims.

BRIEF DESCRIPTION OF THE DRAWINGS

The accompanying drawings, which constitute a part of thisspecification, illustrate several embodiments of the invention andtogether with the description, serve to explain the principles of theinvention.

FIG. 1. Scheme for trans-tympanic antibiotic delivery.

FIGS. 2A-2D. Images of the tympanic membrane (TM) (FIG. 2A) normal,untreated TM; (FIG. 2B) TM with otitis media; (FIG. 2C) TM with gelscontaining ciprofloxacin; (FIG. 2D) TM with gels containingciprofloxacin and permeation enhancers. Space bar=20 μm.

FIG. 3. Graph showing enhanced TM flux from gels containing permeationenhancers. (P407 is poloxamer 407, Cipro=ciprofloxacin, and 3CPE refersto 1% sodium dodecyl sulfate, 0.5% bupivacaine, 2% limonene)

FIG. 4. Graphs showing acoustic brainstem response (ABR) thresholdshifts after application of 18% poloxamer 407 (P407) containing chemicalpermeation enhancers. Horizontal line denotes no change.

FIG. 5. Linear oscillatory shear rheology measurements (100 rads⁻¹, 1%strain, 1° C. min⁻¹) for storage and loss modulus of 18% P407 and 18%P407 with 1% sodium dodecylsulfate, 2% limonene, and 0.5% bupivacaine(3CPE).

FIG. 6. Isobologram showing concentration of CPE B against concentrationof CPE A, and indicating the conditions for synergism and antagonismbetween CPEs.

FIGS. 7A-7B. FIG. 7A. Syntheses of poloxamer-polyphosphoester blockcopolymers. FIG. 7B. Exemplary phosphoester pendant groups formodulating hydrophobicity. FIG. 7C. Exemplary phosphoester groups forenhancing bioadhesiveness.

FIG. 8. Cross-linkable hyaluronic acids (HA) derivatives. Top: HAaldehyde derivative. Bottom: HA hydrazide derivative.

FIG. 9 FTIR for P407-PBP, a poloxamer 407-poly(butoxy)phosphoester, andstarting material P407. The additional stretches indicating addition ofbutoxyphosphoester monomers are indicated.

FIG. 10. Linear oscillatory shear rheology measurements (100 rads⁻¹, 1%strain, 1° C. min⁻¹) for storage and loss modulus of 18% P407-PBP alone(Polymer), with 1% sodium dodecyl sulfate (SDS), with 2% limonene (LIM),with 0.5% bupivacaine (Bup), or with 1% sodium dodecylsulfate, 2%limonene, and 0.5% bupivacaine (3CPE).

FIGS. 11A-11B. P407-PBP with n-butyl groups and DP of 5. FIG. 11A.Chemical structure of the P407-polybutylphosphoester with n-butyl sidegroups; and FTIR spectra of P407 and P407-PBP. FIG. 11B. Chemicalstructure of the P407-polybutylphosphoester with s-butyl side groups andcorresponding FTIR spectra

FIGS. 12A-12B. FIG. 12A. Nuclear magnetic resonance (NMR) of pentablockcopolymers P407-PBP. FIG. 12B. Effect of n- or s-butylphosphoester anddegree of polymerization on storage and loss shear moduli of 18% aqueoussolutions of P407 derivatives, as a function of temperature. Polymerstested include: P407-PBP, with n- or s-butyl groups, and degree ofpolymerization (DP) of 2.5 or 5. Data are means±SD, n=4.

FIGS. 13A-13D. Gelation of aqueous solutions of 18% [P407]. FIG. 13A,Gelation of aqueous solutions of 18% [P407] without CPE; FIG. 13B,3CPE-18% [P407]; FIG. 13C 18% [P407-PBP], and FIG. 13D, 3CPE-18%[P407-PBP], as a function of temperature. Note: 3CPE=2% limonene, 1%SDS, and 0.5% bupivacaine. Data are means±SD, n=4.

FIG. 14. Schematic of the gelation of P407-PBP aqueous solution,facilitated by hydrophobic interactions of polyphosphoester end groupsat elevated temperature (23° C.)

FIGS. 15A-15D. Effects of individual CPEs and polymer concentrations onformulation rheology. FIG. 15A. Effect of individual CPEs on gelationtemperature. FIG. 15B. Effect of individual CPEs on the storage and lossshear moduli of Cip-18% [P407-PBP] solutions at 37° C. FIG. 15C. Effectof P407-PBP concentration on gelation temperature. All formulationscontain 3CPE. FIG. 15D. Effect of P407-PBP concentration on storage andloss shear moduli at 37° C. (from the dotted line in FIG. 15B. Data aremeans±SD, n=4.

FIG. 16. Ex vivo transfer of ciprofloxacin across the TM into areceiving chamber. The hydrogel formulation Cip-3CPE-18% [P407-PBP]enables high cross-TM drug flux. Data are mean±SD, n=4.

FIGS. 17A-17B. Representative photomicrographs of Cip-3CPE-12%[P407-PBP] in normal and diseased chinchilla ears. FIG. 17A. Hematoxylinand eosin (H&E)-stained sections of TM cross-sections in healthy TMs andof TMs after 7 days of OM, without or after treatment with Cip-3CPE-12%[P407-PBP]. Scale bar represents 12 μm. FIG. 17B. H&E-stainedcross-sections of the umbo-malleus region after 7 days of OM, without orafter treatment with Cip-3CPE-12% [P407-PBP]. Scale bar represents 20μm.

FIGS. 18A-18D. In vivo efficacy, pharmacokinetics, impact on hearingsensitivity, and effect on tissue for Cip-3CPE-12% [P407-PBP]. FIG. 18A.Percentage of animals with OM (defined as non-zero cfu values in theirmiddle ear fluid aspirates) before (Day 0) and after receivingCip-3CPE-12% [P407-PBP] (n=10), Cip-3CPE-18% [P407] (n=5), 1%ciprofloxacin ear drop (n=8), or no treatment (n=10). Day 0 reflectsstatus immediately prior to administration of therapeutics. *p=0.0065 byFisher's exact test. FIG. 18B. The time course of bacterial colonyforming units (cfu) from middle ear fluid from animals with OM from NTHitreated with Cip (n=4), Cip-3CPE-18% [P407] (n=5), Cip-3CPE-12%[P407-PBP] (n=10), or no treatment (n=10). Data are means±SD. (Log 10cfu is set to zero instead of minus infinity for the purpose of thisillustration.). FIG. 18C. Concentration of ciprofloxacin over time inthe middle ear fluid of the same animals as in FIG. 18A. The blackdotted line indicates the MIC for NTHi. Inset is magnified drugconcentration range of 0-10 μg/mL. Data are means±SD. FIG. 18D. Shiftsin ABR thresholds in response to acoustic clicks and brief (8 ms) tonebursts of varied frequencies. All data here had the threshold medianprior to the treatment subtracted from them. The red lines indicate theinterquartile range of values prior to treatment (n=8). Measurementsfollowing application of 200 μL of Cip-3CPE-12% [P407-PBP] formulationare in black (n=8): Black boxes and the lines within indicate theinterquartile ranges and medians respectively. Small black squaresindicate the means, and crosses indicate the range.

FIG. 19. Cumulative release of ciprofloxacin at 37° C. from Cip and fromCip-18% [P407], Cip-3CPE-18% [P407], Cip-18% [P407-PBP], andCip-3CPE-18% [P407-PBP] under infinite sink conditions. 2 mgciprofloxacin were contained in each gel and solution at time zero. Dataare means±SD, n=4.

FIGS. 20A-20C. Cytotoxicity of 12% [P407-PBP] and Cip-3CPE-12%[P407-PBP]. FIG. 20A. Survival rates (determined by MTS assay) of humanfibroblasts (hFB), PC12 cells, and keratinocytes, after incubating with12% [P407-PBP] and Cip-3CPE-12% [P407-PBP] for 1 or 3 days. Data aremeans±SD (n=4). FIG. 20B. Live/dead assay of hFB, done to confirm thedata in FIG. 20A by a different assay, after incubation for 1 or 3 dayswith 12% [P407-PBP] or Cip-3CPE-12% [P407-PBP]. Green (GFP) indicateslive cells and red (TRITC) indicates dead cells.

FIG. 20C. hFB cell survival rates were obtained by counting live anddead cells in FIG. 20B and calculating % cell survival=live cells/(livecells+dead cells). Cell counting was done using ImageJ. Data aremeans±SD (n=4). For all images, pair-sample t-test was applied.

FIGS. 21A-21B. FIG. 21A. Permeation enhancement effect of individualCPEs. All curves show the cumulative amount of ciprofloxacin permeatedacross the TM over time. All solutions are made with 12% P407-PBP, 1%Ciprofloxacin, and 1% CPE except the group ‘no CPE’ which does notcontain CPEs. FIG. 21B. Permeation enhancement of effect combinations ofCPEs. All curves show the cumulative amount of ciprofloxacin permeatedacross the TM over time. All solutions are made with 12% P407-PBP, 1%Ciprofloxacin. In particular, stearyl methacrylate (SM) and SDS andbupivacaine (BUP)=1.5% SM (solubility limit), 1% SDS, 0.5% BUP; BP andSDS and BUP=1.5% BP (solubility limit), 1% SDS, 0.5% BUP; LIM and SDSand BUP=2% LIM, 1% SDS, 0.5% BUP.

FIGS. 22A-22C. FIG. 22A. Dose-response curves of SDS, LIM and BUP. Thecumulative amount of ciprofloxacin that permeated across the TM after 48hours increases with the concentration of the CPE in the formulation.All formulations were prepared with: 12% P407-PBP, 4% Ciprofloxacin, andcorresponding concentrations (x-axis) of the individual CPEs. Thecolored dots demonstrate the permeation when two CPEs are added. Forexample, the red dot in the SDS plot represents the permeation when 1%BUP+1% SDS are added to the formulation, instead of 2% SDS. FIG. 22B.Trans-tympanic permeation of levofloxacin formulations in comparisonwith ciprofloxacin formulations. Levofloxacin free drug solution=1.5%Levofloxacin aqueous solution; Levo, P407-PBP+3CPEs=1.5% Levofloxacin,12% P407-PBP, 1% SDS, 2% LIM, 0.5% BUP; Cipro, P407-PBP+3CPEs=1%Ciprofloxacin, 12% P407-PBP, 1% SDS, 2% LIM, 0.5% BUP; Cipro free drugsolution=1% Ciprofloxacin aqueous solution. FIG. 22C. Permeation ofciprofloxacin (left) and dexamethasone (right) across the TM over time.4% Cip-0.1% Dex=4% ciprofloxacin and 0.1% dexamethasone aqueoussolution; 4% Cip-0.1% Dex-3CPE=4% ciprofloxacin, 0.1% dexamethasone, 1%SDS, 2% LIM, 0.5% BUP; 4% Cip-0.1% Dex-3CPE-18% [P407]=4% ciprofloxacin,0.1% dexamethasone, 1% SDS, 2% LIM, 0.5% BUP, 18% P407.

FIGS. 23A-23C. FIG. 23A. Streptococcus pneumoniae (SP) cure rate,showing change in percentage of infected ears over time, for 4%Ciproflaxin formulation with either Ciproflaxin-3CPE orCiproflaxin-3CPE-12% [P407-PBP]. FIG. 23B. Middle ear fluid (MEF)Ciproflaxin concentration over time for various concentrations ofCiproflaxin-3CPE-12% [P407-PBP] and 1% Ciproflaxin-3CPE. FIG. 23C. Exvivo permeation data for formulation of 18% poloxamer 407 (P407) forvarious concentrations of Ciproflaxin-3CPE-12% [P407-PBP] and 4%Ciproflaxin-18% [P407].

DETAILED DESCRIPTION OF CERTAIN EMBODIMENTS

Provided herein are compositions and methods for administering atherapeutic agent to a subject through a barrier. In some embodiments,the composition is for administering a therapeutic agent to the ear of asubject, and the barrier is a tympanic membrane. The compositions andmethods provide for the efficient delivery of the agent to the middleand/or inner ear of the subject. In one aspect, the compositioncomprises a combination of a permeation enhancer, a therapeutic agent,and a matrix forming agent. The permeation enhancer increases the fluxof the therapeutic agent across the barrier (e.g., tympanic membrane),compared to the flux for a composition lacking the permeation enhancer.In various aspects, the composition is a single application compositionfor localized, sustained delivery of a therapeutic agent across thetympanic membrane. In various aspects, the composition is a multipleapplication composition for localized, sustained delivery of atherapeutic agent across the tympanic membrane. The inventivecompositions and methods are particularly useful in treating otitismedia by providing sustained release and delivery of an antibiotic tothe middle ear.

In one aspect, provided herein are compositions comprising:

-   -   (a) a therapeutic agent or a combination of therapeutic agents;    -   (b) a permeation enhancer or a combination of permeation        enhancers, wherein the permeation enhancer or combination of        permeation enhancers increases the flux of flux of the        therapeutic agent or combination of therapeutic agents across a        barrier; and    -   (c) a matrix forming agent or a combination of matrix forming        agents, wherein the matrix forming agent or combination of        matrix forming agents comprises a polymer;        wherein:

the composition forms a gel at temperatures above a phase transitiontemperature;

the phase transition temperature is less than about 37° C.; and

and at least one of conditions (i), (ii), and (iii) are met:

-   -   (i) the phase transition temperature of the composition is less        than the phase transition temperature of a reference composition        plus about 5° C.;    -   (ii) the storage modulus of the composition is greater than        about 15% of the storage modulus of the reference composition at        a temperature of about 37° C.; and    -   (iii) the loss modulus of the composition is between about 80%        and about 120% of the loss modulus of the reference composition        at a temperature of about 37° C.;        wherein the reference composition is the composition in the        absence of (b) the permeation enhancer or combination of        permeation enhancers.

In another aspect, provided herein are compositions comprising:

-   -   (a) a therapeutic agent or a combination of therapeutic agents;    -   (b) a permeation enhancer or a combination of permeation        enhancers, wherein the permeation enhancer or combination of        permeation enhancers increases the flux of flux of the        therapeutic agent or combination of therapeutic agents across a        barrier; and    -   (c) a matrix forming agent or a combination of matrix forming        agents, wherein the matrix forming agent or combination of        matrix forming agents comprises a polymer;        wherein:

the composition forms a gel at temperatures above a phase transitiontemperature;

the phase transition temperature is less than about 37° C.; and

and at least one of conditions (i), (ii), and (iii) are met:

-   -   (i) the phase transition temperature of the composition is less        than the phase transition temperature of a reference composition        plus about 5° C.;    -   (ii) the storage modulus of the composition is greater than        about 15% of the storage modulus of the reference composition or        greater than about 500 Pa, whichever is smaller, at a        temperature of about 37° C.; and    -   (iii) the loss modulus of the composition is between about 15%        and about 150% of the loss modulus of the reference composition        at a temperature of about 37° C.;        wherein the reference composition is the composition in the        absence of (b) the permeation enhancer or combination of        permeation enhancers.

In certain embodiments, condition (i), the phase transition temperatureof the composition is less than the phase transition temperature of thereference composition plus about 5° C., is met. In certain embodiments,condition (ii), the storage modulus of the composition is greater thanabout 15% of the storage modulus of the reference composition, is met.In certain embodiments, condition (ii), the storage modulus of thecomposition is greater than about 15% of the storage modulus of thereference composition, or 500 Pa, whichever is smaller, is met. Incertain embodiments, condition (ii), the storage modulus of thecomposition is greater than about 15% of the storage modulus of thereference composition, or greater than about 1000 Pa, whichever issmaller, is met. In certain embodiments, condition (iii), the lossmodulus of the composition is between about 80% and about 120% of theloss modulus of the reference composition, is met. In certainembodiments, condition (iii), the loss modulus of the composition isbetween about 15% and about 150% of the loss modulus of the referencecomposition, is met. In certain embodiments, both conditions (i) and(ii) are met. In certain embodiments, both conditions (ii) and (iii) aremet. In certain embodiments, both conditions (i) and (iii) are met. Incertain embodiments, each of conditions (i), (ii), and (iii) are met.

In certain embodiments, the therapeutic agent is a single therapeuticagent. In certain embodiments, the therapeutic agent is combination oftwo or more therapeutic agents (e.g., two, three, four). In certainembodiments, the permeation enhancer is a single therapeutic agent. Incertain embodiments, the therapeutic agent is combination of two or moretherapeutic agents (e.g., two, three, four). In certain embodiments, thematrix forming agent is a single matrix forming agent. In certainembodiments, the matrix forming agent is a combination of two or morematrix forming agents (e.g., two, three, four). In certain embodiments,a therapeutic agent or permeation enhancer may act as both a therapeuticagent and a permeation enhancer. In certain embodiments, a therapeuticagent may act as both a therapeutic agent and a permeation enhancer. Incertain embodiments, a permeation enhancer may act as both a therapeuticagent and a permeation enhancer. In certain embodiments, a localanesthetic may act as both a therapeutic agent and a permeationenhancer. In certain embodiments, an amino amide or amino ester localanesthetic may act as both a therapeutic agent and a permeationenhancer. In certain embodiments, an amino amide or amino ester localanesthetic may act as both a therapeutic agent and a permeationenhancer. In certain embodiments, an amino ester local anesthetic mayact as both a therapeutic agent and a permeation enhancer. In certainembodiments, bupivacaine may act as both a therapeutic agent and apermeation enhancer. In certain embodiments, tetracaine may act as botha therapeutic agent and a permeation enhancer.

In certain embodiments, the permeation enhancer or combination ofpermeation enhancers is present in an amount effective to increase theflux of the therapeutic agent across a barrier compared to the referencecomposition (e.g., the composition without the permeation enhancer). Incertain embodiments, the permeation enhancer or combination ofpermeation enhancers is present in an amount effective to increase theflux of the therapeutic agent across a barrier compared to the referencecomposition (e.g., the composition without the permeation enhancer) byat least about 1.05 fold, at least about 1.10 fold, at least about 1.2fold, at least about, at least about 1.3 fold, at least about 1.4 fold,at least about 1.5 fold, at least about 1.6 fold, at least about 1.7fold, at least about 1.8 fold, or at least about 1.9 fold. In certainembodiments, the permeation enhancer or combination of permeationenhancers is present in an amount effective to increase the flux of thetherapeutic agent across a barrier compared to a reference compositionby at least about 2 fold, at least about 2.5 fold, at least about 3fold, at least about 4 fold, at least about 5 fold, at least about 10fold, at least about 25 fold, at least about 50 fold, at least about 100fold, at least about 250 fold, at least about 500 fold, or at leastabout 1000 fold. In certain embodiments, the permeation enhancer orcombination of permeation enhancers is present in an amount effective toincrease the flux of the therapeutic agent across a barrier compared toa reference composition by between about 1.5 fold and about 100 fold.

In certain embodiments, the polymer is a copolymer. In some embodiments,the polymer is biodegradable. In certain embodiments, the copolymer is ablock copolymer. In certain embodiments the copolymer comprises at leastone block of hydrophobic monomers. In certain embodiments, the copolymeris biodegradable, or contains at least one biodegradable block.

As used herein “hydrophobic” refers to a polymer which tends to notdissolve in water and is fat soluble. As used herein “a high degree ofhydrophobicity” refers to a polymer which has a low water watersolubility and that has a high degree of fat solubility. In someembodiments, a hydrophobic polymer comprises hydrophobic side-chains. Insome embodiments, a polymer with a high degree of hydrophobicitycomprises hydrophobic side-chains. Hydrophobic side-chains include butare not limited to, side-chains comprising hydrocarbon radicals, such asalkyl (e.g., methyl), alkenyl, alkynyl, carbocyclyl and aryl.Hydrophobic moieties may also include groups selected from heteroalkyl,heteroalkenyl, heteroalkynyl, heterocyclyl, and heteroaryl, wherein theheteroatom containing group is substantially similar to a hydrocarbongroup (e.g., only 1 or 2 carbons is replaced with a heteroatom).Hydrophobic side-chains may contain groups that are the same as or arederivatives of the side chains of hydrophobic amino acids, including butnot limited to, glycine, alanine, valine, leucine, isoleucine,methionine, phenylalanine, amino isobutyric acid, alloisoleucine,tyrosine, and tryptophan. A non-hydrophobic or hydrophilic polymer is apolymer that tends to dissolve in water.

In certain embodiments, the polymer or copolymer comprises a vinylicpolymer (e.g., PE, PVC, PVDC, PS), a polyacrylate (e.g., polyacrylicacid polymethacrylic acid), a polyether (e.g., PEO, PPO, POM), afluoropolymer (e.g., PTFE), a polysiloxane (e.g., PDMS), apolysaccharide (e.g., cellulose, dextran, hyaluronic acid, chitosan), apolyester (e.g, PET, a polyhydroxyalkanoate (e.g., PHB)), a polyamide(e.g., poly(lactic acid), poly(glycolic acid)), a polyphosphoester, apolyurethane, or a polycarbonate, or copolymers of combinations thereof.In certain embodiments, the copolymer comprises a natural polymer. Insome embodiments, the copolymer comprises a polysaccharide,proteoglycan, glycosaminoglycan, collagen, fibrin, gelatin, or aderivative thereof, or copolymers of combinations thereof.

Exemplary polymer types suitable for the polymer or copolymer include,but are not limited to: polyethers (e.g., polyethylene oxide,polypropylene oxide, polyethylene oxide-polypropylene oxideco-polymers), poloxamers, poloxamines, celluloses and hemicelluloses(e.g., methylcellulose, hydroxypropyl methylcellulose,ethyl(hydroxyethyl) cellulose, xlyoglucan), acetates, phthalates,latexes, polyacrylates (e.g., polyacrylica acid), N-alkylacrylamides(e.g., poly(N-isoproylacrylamide), hyaluronic acids, chitosan, dextranand gellan gum, and derivatives thereof. In some embodiments, thepolymer or copolymer comprises polyethylene oxide or polypropyleneoxide. In some embodiments, the copolymer comprises apolyethylene/polypropylene copolymer or polyethylene/polypropylene blockcopolymer. In some embodiments, the copolymer comprises a poloxamer. Insome embodiments, the copolymer comprises poloxamer 407, poloxamer 188,poloxalene, poloxamer 124, poloxamer 237, or poloxamer 338. In someembodiments, the copolymer comprises poloxamer 407. In certainembodiments, the polymer or copolymer comprises phosphoester monomers.In certain embodiments, the copolymer comprises a poloxamer andphosphoester monomers.

Additional exemplary polymers suitable for the polymer or copolymerinclude aliphatic polyesters such as poly(lactic acid), poly(glycolicacid) and poly(lactic acid-co-glycolic acid); poly(trimethylenecarbonate); polydioxanone and copolymers; poly(butylenes succinate)(such as polybutylene succinate/adipate copolymers, sold as BIONOLLE®,Showa Highpolymer Co. Ltd.) and poly(butylene adipate); polyanhydrides,such as poly(adipic anhydride) and poly(sebacicacid-co-1,3-bis(p-carboxyphenoxy) carboxyphenoxy) propane; poly(orthoester)s; poly(ester amide)s, such as polymers based on 1,4-butanediol,adipic acid, and 1,6-aminohexanoic acid (BAK1095, Bayer AG, Leverkusen);poly(ester urethane)s; poly(ester anhydride)s; poly(ester carbonate)s,such as tyrosine-poly (alkylene oxide)-derived poly(ether carbonate)s;polyphosphazenes, polyarylates, such as tyrosine-derived polyacrylates;poly(ether ester)s, such as poly(butylene terephthalate)-poly(ethyleneglycol) copolymers (PolyActiveo®), poly(ε-caprolactone)-poly(ethyleneglycol)) block copolymers and poly(ethylene oxide)-poly(hydroxybutyrate) block copolymers; polypropylfumerates; polyacetals;polyethers; biodegradable polycyanoacrylates; biodegradablepolyurethanes; polyphosphoesters; poly(amide-enamines); polyamides;poly(amino acids); polycaprolactones; and polyhydroxyalkanoates.

In certain embodiments, the polymer or copolymer comprises poly (lacticacid) (PLA), poly (glycolic acid) (PGA), a copolymers of PLA and PGA,(e.g., poly (lactide-co-glycolide) (PLG)), poly (caprolactone) (PCL),poly (lactide-co-caprolactone) (PLC), or poly(glycolide-co-caprolactone) (PGC).

In certain embodiments, the copolymer is a block copolymer of formulaA-B-A, wherein B is a hydrophobic block and each A is a non-hydrophobicblocks. In certain embodiments, the copolymer is a block copolymer offormula C-A-B-A-C, wherein each B or C is a hydrophobic blocks, and Aare non-hydrophobic blocks. Polymers A-B-A and C-A-B-A-C may alsocomprise terminal groups attached to end block A or C. In certainembodiments, B and C are different polymers, In certain embodiments, Band C are the same polymer. In certain embodiments, each block A is apolymer of between 1 and 400 monomers. In certain embodiments, block Ais a polymer of between 20 and 200 monomers. In certain embodiments eachblock B, is a polymer of between 1 and 400 monomers. In certainembodiments, block B is a polymer of between 20 and 200 monomers. Incertain embodiments each block C, is a polymer of between 1 and 400monomers. In certain embodiments, block C is a polymer of between 20 and200 monomers. In certain embodiments, each block A comprises a singletype of monomer. In certain embodiments, each block A comprises morethan one type of monomer. In certain embodiments, each block B comprisesa single type of monomer. In certain embodiments, each block B comprisesmore than one type of monomer. In certain embodiments, each block Ccomprises a single type of monomer. In certain embodiments, each block Ccomprises more than one type of monomer.

In certain embodiments, polymer A is a hydrophilic polyether (e.g.,polyethylene oxide). In certain embodiments, polymer A is a hydrophilicpolyester (e.g., polyglycolic acid). In certain embodiments, polymer Bis a hydrophobic polyether (e.g., polypropylene oxide). In certainembodiments, polymer B is a hydrophobic polyester (e.g., polylacticacid). In certain embodiments, polymer B is a hydrophobic polyether(e.g., polypropylene oxide). In certain embodiments, polymer B is ahydrophobic polyester (e.g., polylactic acid). In certain embodiments,polymer C is a polyphosphoester.

The composition may be a liquid prior to warming above the phasetransition temperature. In some embodiments, the phase transitiontemperature is at or below the body temperature of a subject (e.g.,about 37° C.). Thus, the composition may form a gel when administered toa subject, e.g., when the composition contacts a biological surface. Insome embodiments, the phase transition temperature is between about 0°C. and about 37° C., between about 10° C. and about 37° C., betweenabout 15° C. and about 37° C., between about 20° C. and about 37° C.,between about 25° C. between about 30° C. and about 37° C., betweenabout 30° C. and about 35° C., or between about 35° C. and about 40° C.In some embodiments, the phase transition temperature is between about20° C. and about 37° C. In some embodiments, the phase transitiontemperature is between about 0° C. and about 60° C., between about 10°C. and about 50° C., between about 20° C. and about 40° C., or betweenabout 25° C. and about 35° C. In some embodiments, the phase transitiontemperature is between about 20° C. and 25° C., between about 25° C. andabout 30° C., between about 30° C. and about 35° C., or between about35° C. and about 40° C. In some embodiments, the phase transitiontemperature is between about 10° C. and about 50° C. In someembodiments, the phase transition temperature is between about 20° C.and about 40° C. In some embodiments, the phase transition temperatureis between about 15° C. and about 40° C.

For any composition comprising a matrix forming agent, the phasetransition temperature of the composition may change if an additive isadded to the composition. The phase transition temperature of acomposition with an additive versus a reference composition without theadditive may be higher, lower, or the same depending on characteristicsof the composition and the additive. The term reference composition asused herein, refers to a composition which contains the same componentsas the composition to which it is being compared, with the exception ofa specified component (e.g., the permeation enhancer). Unless otherwisestated, the difference in % weight/volume from including or excludingthe permeation enhancer is made up by a change in % weight/volume of thesolvent (e.g., water). In certain embodiments, the reference compositioncomprises the therapeutic agent and the matrix forming agent, but notthe permeation enhancer. In certain embodiments, the referencecomposition comprises the matrix forming agent, but not the therapeuticagent or the permeation enhancer. In certain embodiments, the referencecomposition comprises the permeation enhancer and the matrix formingagent, but not the therapeutic agent.

In certain embodiments, the phase transition temperature of thecomposition is greater than the phase transition temperature of thereference composition (e.g., the composition without the permeationenhancer). In certain embodiments, the phase transition temperature ofthe composition is less than the phase transition temperature of thereference composition (e.g., the composition without the permeationenhancer) plus about 5° C., 4° C., about 3° C., about 2° C., or about 1°C. In certain embodiments, the phase transition temperature of thecomposition is less than the phase transition temperature of thereference composition (e.g., the composition without the permeationenhancer) plus about 5° C. In certain embodiments, the phase transitiontemperature of the composition is less than the phase transitiontemperature of the reference composition (e.g., the composition withoutthe permeation enhancer) plus about 37° C. In certain embodiments, thephase transition temperature of the composition is less than the phasetransition temperature of the reference composition (e.g., thecomposition without the permeation enhancer) plus about 30° C., about20° C., or about 10° C. In certain embodiments, the phase transitiontemperature of the composition is less than the phase transitiontemperature of the reference composition (e.g., the composition withoutthe permeation enhancer).

In certain embodiments, the phase transition temperature of thecomposition is less than the phase transition temperature of thereference composition (e.g., the composition without the permeationenhancer) plus about 5° C., plus about 2° C., or plus about 1° C., andis higher than about 0° C., about 10° C., about 15° C., about 20° C.,about 25° C., or about 30° C. In certain embodiments, the phasetransition temperature of the composition is less than the phasetransition temperature of the reference composition (e.g., thecomposition without the permeation enhancer) plus about 5° C., and ishigher than about 20° C.

As a non-limiting example consider the following compositions. The phasetransition temperature of a composition “A” comprising: (i) 1%ciprofloxacin and (ii) 18% poloxamer 407/polybutylphosphoester copolymeris about 33° C. For the corresponding composition “B” comprising thecomponent of “A” and (iii) 1% sodium dodecyl sulfate, the phasetransition temperature decreases to about 31° C. For the correspondingcomposition “C” comprising the components of “A” and (iii) 0.5%bupivacaine, the phase transition temperature remains about 33° C.Compositions “B” and “C” would both meet the criteria for the phasetransition temperature of the composition with the permeation enhancerbeing less than, or slightly higher (e.g., <5° C.) than, the phasetransition temperature of the composition without the permeationenhancer.

In certain embodiments, the composition is a gel at temperatures abovethe phase transition temperature and below about 60° C., below about 50°C., or below about 40° C. In certain embodiments, the composition is agel at temperatures above the phase transition temperature and belowabout 50° C. In certain embodiments, the composition is a gel attemperatures between about 0° C. and about 60° C., between about 10° C.and about 50° C., between about 20° C. and about 40° C., or betweenabout 25° C. and about 35° C. In some embodiments, the composition is agel is at temperatures between about 20° C. and 25° C., between about25° C. and about 30° C., between about 30° C. and about 35° C., orbetween about 35° C. and about 40° C. In some embodiments, thecomposition is a gel at temperatures between about 10° C. and about 50°C. In some embodiments, the composition is a gel at temperatures betweenabout 20° C. and about 40° C. In some embodiments, the composition is agel at temperatures between about 15° C. and about 40° C.

For any composition comprising a matrix forming agent, the storagemodulus and loss modulus of the composition may change if an additive isadded to the composition. The storage modulus of a composition with anadditive versus the same composition without the additive may be higher,lower, or the same depending on characteristics of the composition andthe additive. The loss modulus of a composition with an additive versusa reference composition without the additive may be higher, lower, orthe same depending on characteristics of the composition and theadditive. In certain embodiments, the reference composition comprisesthe therapeutic agent and the matrix forming agent, but not thepermeation enhancer. In certain embodiments, the reference compositioncomprises the matrix forming agent, but not the therapeutic agent or thepermeation enhancer. In certain embodiments, the reference compositioncomprises the permeation enhancer and the matrix forming agent, but notthe therapeutic agent.

In certain embodiments, condition (ii), the storage modulus of thecomposition is greater than about 15% of the storage modulus of thereference composition, or greater than about 500 Pa, whichever issmaller, is met. In certain embodiments, condition (ii), the storagemodulus of the composition is greater than about 15% of the storagemodulus of the reference composition, or greater than about 1000 Pa,whichever is smaller, is met. In certain embodiments, the storagemodulus of the composition is greater than about 15%, greater than about30%, greater than about 50%, greater than about 60%, greater than about70%, greater than about 80%, greater than about 90%, or greater thanabout 100% of the storage modulus of the reference composition (e.g.,the composition without the permeation enhancer) at a given temperature.In certain embodiments, the storage modulus of the composition isgreater than about 15% of the storage modulus of the referencecomposition (e.g., the composition without the permeation enhancer) at agiven temperature. In certain embodiments, the storage modulus of thecomposition is greater than about 30% of the storage modulus of thereference composition (e.g., the composition without the permeationenhancer) at a given temperature. In certain embodiments, the storagemodulus of the composition is greater than about 50% of the storagemodulus of the reference composition (e.g., the composition without thepermeation enhancer) at a given temperature. In certain embodiments, thestorage modulus of the composition is greater than about 70% of thestorage modulus of the reference composition (e.g., the compositionwithout the permeation enhancer) at a given temperature. In certainembodiments, the storage modulus of the composition is greater thanabout 80% or about 90% of the storage modulus of the referencecomposition (e.g., the composition without the permeation enhancer) at agiven temperature. In certain embodiments, the storage modulus of thecomposition is greater than about 100% of the storage modulus of thereference composition (e.g., the composition without the permeationenhancer) at a given temperature. In certain embodiments, the storagemodulus of the composition is greater than about 110%, greater thanabout 120%, greater than about 130%, greater than about 140%, greaterthan about 150%, greater than about 175%, or greater than about 200% ofthe storage modulus of the reference composition (e.g., the compositionwithout the permeation enhancer) at a given temperature. In certainembodiments, the storage modulus of the composition is less than about200%, less than about 500%, or less than about 1000% of the storagemodulus of the reference composition (e.g., the composition without thepermeation enhancer) at a given temperature. In certain embodiments, thegiven temperature is about 37° C. In certain embodiments, the giventemperature is a temperature between the phase transition temperatureand about 37° C.

In certain embodiments, the loss modulus of the composition is less thanabout 200%, less than about 150%, less than about 125%, less than about110%, or less than about 100% of the storage modulus of the referencecomposition (e.g., the composition without the permeation enhancer) at agiven temperature. In certain embodiments, the loss modulus of thecomposition is greater than about 50%, less than about 75%, or greaterthan about 90% of the loss modulus of the reference composition (e.g.,the composition without the permeation enhancer) at a given temperature.In certain embodiments, the loss modulus of the composition is betweenabout 50%, and about 150%, between about 70%, and about 130%, betweenabout 80%, and about 120%, or between about 90%, and about 110% of theloss modulus of the reference composition (e.g., the composition withoutthe permeation enhancer) at a given temperature. In certain embodiments,the loss modulus of the composition is between about 80%, and about 120%of the loss modulus of the reference composition (e.g., the compositionwithout the permeation enhancer) at a given temperature. In certainembodiments, condition (iii), the loss modulus of the composition isbetween about 15% and about 150% of the loss modulus of the referencecomposition at a temperature of about 37° C. In certain embodiments, thegiven temperature is about 37° C. In certain embodiments, the giventemperature is a temperature between the phase transition temperatureand about 37° C.

In certain embodiments, the composition comprises at least about 0.1%permeation enhancer. In certain embodiments, the composition comprisesat least about 0.5% permeation enhancer. In certain embodiments, thecomposition comprises at least about 1% permeation enhancer. In certainembodiments, the composition comprises at least about 2% permeationenhancer. In certain embodiments, the composition comprises at leastabout 3% permeation enhancer. In certain embodiments, the compositioncomprises at least about 4% permeation enhancer. In certain embodiments,the composition comprises at least about 5% permeation enhancer. Incertain embodiments, the composition comprises at least about 6%, atleast about 7%, at least about 8%, at least about 9%, or at least about10% permeation enhancer. In certain embodiments, the compositioncomprises at least about 0.5% weight per volume composition (wt/vol)permeation enhancer. In certain embodiments, the composition comprisesat least about 1% wt/vol permeation enhancer. In certain embodiments,the composition comprises at least about 2% wt/vol permeation enhancer.In certain embodiments, the composition comprises at least about 3%wt/vol permeation enhancer. In certain embodiments, the compositioncomprises at least about 4% wt/vol permeation enhancer. In certainembodiments, the composition comprises at least about 5% permeationenhancer. In certain embodiments, the composition comprises at leastabout 6% wt/vol permeation enhancer. In certain embodiments, thecomposition comprises at least about 7% wt/vol permeation enhancer. Incertain embodiments, the composition comprises at least about 8% wt/volpermeation enhancer, In certain embodiments, the composition comprisesat between about 0.1% and about 1% permeation enhancer. In certainembodiments, the composition comprises at between about 0.5% and about3% permeation enhancer. In certain embodiments, the compositioncomprises at between about 0.5% and about 10% permeation enhancer. Incertain embodiments, the composition comprises at between about 2% andabout 10% permeation enhancer.

In certain embodiments, the composition is applied to a surface oftemperature equal to or above the phase transition temperature. In someembodiments, the surface is a biological surface. In certainembodiments, the surface is skin. In certain embodiments, the surface isa surface in the ear canal of a subject. In certain embodiments, thesubject is a tympanic membrane. In certain embodiments, the surface is asurface in the respiratory tract of a subject (e.g., in the nasal cavityor buccal cavity). In certain embodiments, the surface is a surface inthe mouth (e.g., surface of teeth or gums) of a subject. The compositionmay be administered to an interior body surface, for example, byintradermal or interdermal delivery or during a surgical procedure. Incertain embodiments, the surface is an intradermal surface. In certainembodiments, the surface is the surface of an organ (e.g., heart, lung,spleen, pancreas, kidney, liver, stomach, intestine, bladder). Incertain embodiments, the surface is connective tissue. In certainembodiments, the surface is muscle tissue (e.g., smooth muscle, skeletalmuscle, cardiac muscle). In certain embodiments, the surface is nervoustissue (e.g., brain, spinal cord). In certain embodiments, the surfaceis epithelial tissue. In certain embodiments, the surface is a surfaceof the alimentary canal (e.g., colon, rectum). In certain embodiments,the surface is epithelial tissue. In certain embodiments, the surface isa surface of the reproductive tract (e.g., vagina, cervix). In certainembodiments, the surface is bone. In certain embodiments, the surface isvascular tissue. In certain embodiments, the surface is a wound bed. Incertain embodiments, the surface is a biofilm. In certain embodiments,the surface is hair or fur. In certain embodiments, the surface is thesurface of a medical implant.

Generally, for addition of a permeation enhancer a small change or nochange in the phase transition temperature, storage modulus, or lossmodulus is preferred. A small change is considered a phase transitiontemperature change of less than 5° C., or a modulus change of less than10%. For changes in the phase transition temperature, a lower phasetransition temperature for the composition with the permeation enhanceris preferred. A shift to a lower phase transition temperature mayreferred to as a ‘left-shift’ or ‘L-shift’ as opposed to a ‘right-shift’or ‘R-shift’. For changes in the storage modulus, a higher storagemodulus for the composition with the permeation enhancer is preferred.For changes in the loss modulus, a lower loss modulus for thecomposition with the permeation enhancer is preferred.

In certain embodiments, the phase transition temperature of thecomposition is within about 5° C., within about 3° C., or within about1° C. of the phase transition temperature of a reference composition,wherein the composition comprises permeation enhancer P1 and thereference composition does not comprise permeation enhancer P1. Incertain embodiments, the storage modulus of the composition is withinabout 10%, within about 5%, or within about 2% ° C. of the storagemodulus of a reference composition, wherein the composition comprisespermeation enhancer P1 and the reference composition does not comprisepermeation enhancer P1. In certain embodiments, the loss modulus of thecomposition is within about 10%, within about 5%, or within about 2% °C. of the loss modulus of a reference composition, wherein thecomposition comprises permeation enhancer P1 and the referencecomposition does not comprise permeation enhancer P1. In certainembodiments, the phase transition temperature of the composition iswithin about 5° C., within about 3° C., or within about 1° C. of thephase transition temperature of a reference composition, and the storagemodulus of the composition is within about 10%, within about 5%, orwithin about 2% ° C. of the storage modulus of the referencecomposition, wherein the composition comprises permeation enhancer P1and the reference composition does not comprise permeation enhancer P1.

In certain embodiments, permeation enhancer P1 is a surfactant (anionic,cationic, nonionic, zwitterionic), terpene, anesthetic, amino amide,amino ester, azide-containing compound, or alcohol. In certainembodiments, permeation enhancer P1 is a surfactant (anionic, cationic,nonionic, zwitterionic), terpene, anesthetic, amino amide, amino ester,azide-containing compound, pyrrolidone, sulfoxide, fatty acid, oralcohol. In certain embodiments, permeation enhancer P1 is a surfactant(e.g., sodium dodecyl sulfate, ammonium lauryl sulfate, sodium laurylsulfate, cetyl trimethylammonium bromide, cetylpyridinium chloride,benzethonium chloride, cocamidopropyl betaine, cetyl alcohol, oleylalcohol, octyl glucoside, decyl maltoside, sodium octyl sulfate, sodiumdecyl sulfate, sodium tetradecyl sulfate, sodium heptadecyl sulfate,sodium eicosyl sulfate, nicotine sulfate, sodium taurocholic sulfate,dimethyl sulfoxide, sodium tridecyl phosphate; decyldimethyl ammoniopropane sulfonate, chembetaine oleyl, myristyldimethyl ammonio propanesulfonate; benzyl pyridinium chloride, dodecyl pyridinium chloride,cetyl pyridinium chloride, benzyldimethyl dodecyl ammonium chloride,benzyldimethyl dodecyl ammonium chloride, benzyldimethyl myristylammonium chloride, benzyldimethyl stearyl ammonium chloride,octyltrimethylammonium bromide, dodecyltrimethylammonium bromide,Polysorbate 20, Polysorbate 40, Polysorbate 60, Polysorbate 80). Incertain embodiments, permeation enhancer P1 is a terpene (e.g.,limonene, cymene, pinene, camphor, menthol, comphone, phellandrine,sabinene, terpinene, borneol, cineole, geraniol, linalol, pipertone,terpineol, eugenol, eugenol acetate, safrole, benzyl benzoate, humulene,beta-caryophylene, eucakytol, hexanoic acid, octanoic acid, decanoicacid, undecanoic acid, dodecanoic acid, tridecanoic acid, myristic acid,palmitic acid, stearic acid, oleic acid, linoleic acid, linolenic acid,cholic acid; ethyl undecanoate, methyl laurate, methyl myristate,isopropyl myristate, isopropyl palmitate, palmityl palmitate, diethylsebaccate, glyceryl monolaurate, glyceryl monooleate, ethylpiperazinecarboxylate). In certain embodiments, permeation enhancer P1 is aterpene. In certain embodiments, the composition comprises between0.5-6.0% terpene by weight. In certain embodiments, the compositioncomprises between 1.5-3.0% terpene by weight. In certain embodiments,the composition comprises between 1.5-2.0% terpene by weight. In certainembodiments, the composition comprises 2.0% terpene by weight. Incertain embodiments, the composition comprises between 1.5-3.0% limoneneby weight. In certain embodiments, the composition comprises between1.5-2.0% limonene by weight. In certain embodiments, the compositioncomprises 2.0% limonene by weight. In certain embodiments, permeationenhancer P1 is an anesthetic (e.g., bupivacaine, tetracaine, procaine,proparacaine, propoxycaine, dimethocaine, cyclomethycaine,chloroprocaine, benzocaine, lidocaine, prilocaine, levobupivicaine,ropivacaine, dibucaine, articaine, carticaine, etidocaine, mepivacaine,piperocaine, trimecaine). In some embodiments, permeation enhancer P1 isbupivacaine. In some embodiments, permeation enhancer P1 is sodiumdodecyl sulfate. In some embodiments, permeation enhancer P1 islimonene. In some embodiments, permeation enhancer P1 is a combinationof at least two of a surfactant, terpene, and anesthetic. In someembodiments, permeation enhancer P1 is a combination of bupivacaine,sodium dodecyl sulfate, and limonene. In certain embodiments, permeationenhancer P1 is sodium lauroyl sarcosinate, sorbitan monooleate,octoxynol-9, diethyl sebacate, sodium polyacrylate (2500000 MW), oroctyldodecanol.

In certain embodiments, the phase transition temperature of thecomposition is less than the phase transition temperature of a referencecomposition, wherein the composition comprises permeation enhancer P2and the reference composition does not comprise permeation enhancer P2.In certain embodiments, the storage modulus of the composition is withinabout 10%, within about 5%, or within about 2% ° C. of the storagemodulus of a reference composition, wherein the composition comprisespermeation enhancer P2 and the reference composition does not comprisepermeation enhancer P2. In certain embodiments, the storage modulus ofthe composition is within about 100%, within about 10%, within about 5%,or within about 2% ° C. of the storage modulus of a referencecomposition, wherein the composition comprises permeation enhancer P2and the reference composition does not comprise permeation enhancer P2.In certain embodiments, the loss modulus of the composition is withinabout 10%, within about 5%, or within about 2% ° C. of the loss modulusof a reference composition, wherein the composition comprises permeationenhancer P2 and the reference composition does not comprise permeationenhancer P2. In certain embodiments, the phase transition temperature ofthe composition is less than the phase transition temperature of areference composition, and the storage modulus of the composition iswithin about 10%, within about 5%, or within about 2% ° C. of thestorage modulus of a reference composition, wherein the compositioncomprises permeation enhancer P2 and the reference composition does notcomprise permeation enhancer P2.

In certain embodiments, permeation enhancer P2 is a surfactant (anionic,cationic, nonionic, zwitterionic), terpene, anesthetic, amino amide,amino ester, azide-containing compound, or alcohol. In certainembodiments, permeation enhancer P2 is a surfactant (anionic, cationic,nonionic, zwitterionic), terpene, anesthetic, amino amide, amino ester,azide-containing compound, pyrrolidone, sulfoxide, fatty acid, oralcohol. In certain embodiments, permeation enhancer P2 is a surfactant(e.g., sodium dodecyl sulfate, ammonium lauryl sulfate, sodium laurylsulfate, cetyl trimethylammonium bromide, cetylpyridinium chloride,benzethonium chloride, cocamidopropyl betaine, cetyl alcohol, oleylalcohol, octyl glucoside, decyl maltoside, sodium octyl sulfate, sodiumdecyl sulfate, sodium tetradecyl sulfate, sodium heptadecyl sulfate,sodium eicosyl sulfate, nicotine sulfate, sodium taurocholic sulfate,dimethyl sulfoxide, sodium tridecyl phosphate; decyldimethyl ammoniopropane sulfonate, chembetaine oleyl, myristyldimethyl ammonio propanesulfonate; benzyl pyridinium chloride, dodecyl pyridinium chloride,cetyl pyridinium chloride, benzyldimethyl dodecyl ammonium chloride,benzyldimethyl dodecyl ammonium chloride, benzyldimethyl myristylammonium chloride, benzyldimethyl stearyl ammonium chloride,octyltrimethylammonium bromide, dodecyltrimethylammonium bromide,Polysorbate 20, Polysorbate 40, Polysorbate 60, Polysorbate 80). Incertain embodiments, permeation enhancer P2 is a terpene (e.g.,limonene, cymene, pinene, camphor, menthol, comphone, phellandrine,sabinene, terpinene, borneol, cineole, geraniol, linalol, pipertone,terpineol, eugenol, eugenol acetate, safrole, benzyl benzoate, humulene,beta-caryophylene, eucakytol, hexanoic acid, octanoic acid, decanoicacid, undecanoic acid, dodecanoic acid, tridecanoic acid, myristic acid,palmitic acid, stearic acid, oleic acid, linoleic acid, linolenic acid,cholic acid; ethyl undecanoate, methyl laurate, methyl myristate,isopropyl myristate, isopropyl palmitate, palmityl palmitate, diethylsebaccate, glyceryl monolaurate, glyceryl monooleate, ethylpiperazinecarboxylate). In certain embodiments, permeation enhancer P2 is aterpene. In certain embodiments, the composition comprises between0.5-6.0% terpene by weight. In certain embodiments, the compositioncomprises between 1.5-3.0% terpene by weight. In certain embodiments,the composition comprises between 1.5-2.0% terpene by weight. In certainembodiments, the composition comprises 2.0% terpene by weight. Incertain embodiments, the composition comprises between 1.5-3.0% limoneneby weight. In certain embodiments, the composition comprises between1.5-2.0% limonene by weight. In certain embodiments, the compositioncomprises 2.0% limonene by weight. In certain embodiments, permeationenhancer P2 is an anesthetic (e.g., bupivacaine, tetracaine, procaine,proparacaine, propoxycaine, dimethocaine, cyclomethycaine,chloroprocaine, benzocaine, lidocaine, prilocaine, levobupivicaine,ropivacaine, dibucaine, articaine, carticaine, etidocaine, mepivacaine,piperocaine, trimecaine). In some embodiments, permeation enhancer P2 isbupivacaine. In some embodiments, permeation enhancer P2 is sodiumdodecyl sulfate. In some embodiments, permeation enhancer P2 islimonene. In some embodiments, permeation enhancer P2 is a combinationof at least two of a surfactant, terpene, and anesthetic. In someembodiments, permeation enhancer P2 is a combination of bupivacaine,sodium dodecyl sulfate, and limonene. In certain embodiments, permeationenhancer P2 is sodium lauroyl sarcosinate, sorbitan monooleate,octoxynol-9, diethyl sebacate, sodium polyacrylate (2500000 MW), oroctyldodecanol.

In certain embodiments, the phase transition temperature of thecomposition is less than the phase transition temperature of a referencecomposition, wherein the composition comprises permeation enhancer P3and the reference composition does not comprise permeation enhancer P3.In certain embodiments, the storage modulus of the composition isgreater than the storage modulus of a reference composition, wherein thecomposition comprises permeation enhancer P3 and the referencecomposition does not comprise permeation enhancer P3. In certainembodiments, the loss modulus of the composition is greater than theloss modulus of a reference composition, wherein the compositioncomprises permeation enhancer P3 and the reference composition does notcomprise permeation enhancer P3. In certain embodiments, the phasetransition temperature of the composition is less than the phasetransition temperature of a reference composition, and the storagemodulus of the composition is greater than the storage modulus of areference composition, wherein the composition comprises permeationenhancer P3 and the reference composition does not comprise permeationenhancer P3.

In certain embodiments, permeation enhancer P3 is a surfactant (anionic,cationic, nonionic, zwitterionic), terpene, anesthetic, amino amide,amino ester, azide-containing compound, or alcohol. In certainembodiments, permeation enhancer P3 is a surfactant (anionic, cationic,nonionic, zwitterionic), terpene, anesthetic, amino amide, amino ester,azide-containing compound, pyrrolidone, sulfoxide, fatty acid, oralcohol. In certain embodiments, permeation enhancer P3 is a surfactant(e.g., sodium dodecyl sulfate, ammonium lauryl sulfate, sodium laurylsulfate, cetyl trimethylammonium bromide, cetylpyridinium chloride,benzethonium chloride, cocamidopropyl betaine, cetyl alcohol, oleylalcohol, octyl glucoside, decyl maltoside, sodium octyl sulfate, sodiumdecyl sulfate, sodium tetradecyl sulfate, sodium heptadecyl sulfate,sodium eicosyl sulfate, nicotine sulfate, sodium taurocholic sulfate,dimethyl sulfoxide, sodium tridecyl phosphate; decyldimethyl ammoniopropane sulfonate, chembetaine oleyl, myristyldimethyl ammonio propanesulfonate; benzyl pyridinium chloride, dodecyl pyridinium chloride,cetyl pyridinium chloride, benzyldimethyl dodecyl ammonium chloride,benzyldimethyl dodecyl ammonium chloride, benzyldimethyl myristylammonium chloride, benzyldimethyl stearyl ammonium chloride,octyltrimethylammonium bromide, dodecyltrimethylammonium bromide,Polysorbate 20, Polysorbate 40, Polysorbate 60, Polysorbate 80). Incertain embodiments, permeation enhancer P3 is a terpene (e.g.,limonene, cymene, pinene, camphor, menthol, comphone, phellandrine,sabinene, terpinene, borneol, cineole, geraniol, linalol, pipertone,terpineol, eugenol, eugenol acetate, safrole, benzyl benzoate, humulene,beta-caryophylene, eucakytol, hexanoic acid, octanoic acid, decanoicacid, undecanoic acid, dodecanoic acid, tridecanoic acid, myristic acid,palmitic acid, stearic acid, oleic acid, linoleic acid, linolenic acid,cholic acid; ethyl undecanoate, methyl laurate, methyl myristate,isopropyl myristate, isopropyl palmitate, palmityl palmitate, diethylsebaccate, glyceryl monolaurate, glyceryl monooleate, ethylpiperazinecarboxylate). In certain embodiments, permeation enhancer P3 is aterpene. In certain embodiments, the composition comprises between0.5-6.0% terpene by weight. In certain embodiments, the compositioncomprises between 1.5-3.0% terpene by weight. In certain embodiments,the composition comprises between 1.5-2.0% terpene by weight. In certainembodiments, the composition comprises 2.0% terpene by weight. Incertain embodiments, the composition comprises between 1.5-3.0% limoneneby weight. In certain embodiments, the composition comprises between1.5-2.0% limonene by weight. In certain embodiments, the compositioncomprises 2.0% limonene by weight. In certain embodiments, permeationenhancer P3 is an anesthetic (e.g., bupivacaine, tetracaine, procaine,proparacaine, propoxycaine, dimethocaine, cyclomethycaine,chloroprocaine, benzocaine, lidocaine, prilocaine, levobupivicaine,ropivacaine, dibucaine, articaine, carticaine, etidocaine, mepivacaine,piperocaine, trimecaine). In some embodiments, permeation enhancer P3 isbupivacaine. In some embodiments, permeation enhancer P3 is sodiumdodecyl sulfate. In some embodiments, permeation enhancer P3 islimonene. In some embodiments, permeation enhancer P3 is a combinationof at least two of a surfactant, terpene, and anesthetic. In someembodiments, permeation enhancer P3 is a combination of bupivacaine,sodium dodecyl sulfate, and limonene. In certain embodiments, permeationenhancer P3 is sodium lauroyl sarcosinate, sorbitan monooleate,octoxynol-9, diethyl sebacate, sodium polyacrylate (2500000 MW), oroctyldodecanol.

In certain embodiments, the composition is useful in treating a disease.In some embodiments, the composition is useful in treating an infectiousdisease. In some embodiments, the composition is useful in treating anear disease (e.g., the barrier is the tympanic membrane). In someembodiments, the composition is useful in treating otitis media.

As described, the gelation temperature (phase transition temperature) ofthe composition is one factor in determining whether the suitability ofthe composition (e.g., to allow for sustained delivery to the tympanicmembrane). The temperature at which the storage modulus exceeds the lossmodulus is considered the gelation temperature. Compositions herein mayhave a gelation temperature lower or higher than 37° C., but preferablylower than 37° C. to accelerate gelation right after administration uponexposure of the composition, in particular the matrix forming agent, tobody heat.

The timing of the sol-gel transition will impact the ease ofadministration. In general a faster in situ transition is useful foradministration to subjects (e.g., children resisting compliance). Incertain embodiments, the composition gels within about 5 s, about 10 s,about 20 s, about 30 s, about 1 minute, about 5 minutes, or about 10minutes of administration (e.g., to the ear canal). In some embodiments,the composition gels in the range of about 1 s to about 20 s afteradministration.

In certain embodiments, the composition is stored cold (e.g.,refrigerated at about 5° C.) prior to administration. Cold storage maybe useful for compositions with gelation temperatures below roomtemperature to prevent gelation prior to administration or duringhandling.

In one aspect, provided herein are compositions comprising:

-   -   (a) a therapeutic agent or a combination of therapeutic agents;    -   (b) a permeation enhancer or a combination of permeation        enhancers, wherein the permeation enhancer or combination of        permeation enhancers increases the flux of the therapeutic agent        or combination of therapeutic agents across a barrier; and    -   (c) a matrix forming agent or a combination of matrix forming        agents, wherein the matrix forming agent or combination of        matrix forming agents comprises a block copolymer containing        hydrophobic monomers (e.g., phosphoester monomers);        wherein:

the composition forms a gel at temperatures above a phase transitiontemperature; and

the phase transition temperature is less than about 37° C.;

and at least one of conditions (i), (ii), and (iii) are met:

-   -   (i) the phase transition temperature of the composition is less        than the phase transition temperature of a reference composition        plus about 5° C.;    -   (ii) the storage modulus of the composition is greater than        about 70% of the storage modulus of the reference composition at        a temperature of about 37° C.; and    -   (iii) the loss modulus of the composition is between about 80%        and about 120% of the loss modulus of the reference composition        at a temperature of about 37° C.;        wherein the reference composition is the composition in the        absence of the permeation enhancer or combination of permeation        enhancers.

In one aspect, provided herein are compositions comprising:

-   -   (a) a therapeutic agent or a combination of therapeutic agents;    -   (b) a permeation enhancer or a combination of permeation        enhancers, wherein the permeation enhancer or combination of        permeation enhancers increases the flux of the therapeutic agent        or combination of therapeutic agents across a barrier; and    -   (c) a matrix forming agent or a combination of matrix forming        agents, wherein the matrix forming agent or combination of        matrix forming agents comprises a block copolymer containing        hydrophobic monomers (e.g., phosphoester monomers);        wherein:

the composition forms a gel at temperatures above a phase transitiontemperature; and

the phase transition temperature is less than about 37° C.;

and at least one of conditions (i), (ii), and (iii) are met:

-   -   (i) the phase transition temperature of the composition is less        than the phase transition temperature of a reference composition        plus about 5° C.;    -   (ii) the storage modulus of the composition is greater than        about 70% of the storage modulus of the reference composition at        a temperature of about 37° C.; and    -   (iii) the loss modulus of the composition is between about 15%        and about 150% of the loss modulus of the reference composition        at a temperature of about 37° C.;

wherein the reference composition is the composition in the absence ofthe permeation enhancer or combination of permeation enhancers.

In another aspect, provided herein are compositions for treating aninfectious disease comprising:

-   -   (a) a therapeutic agent or a combination of therapeutic agents;    -   (b) a permeation enhancer or a combination of permeation        enhancers, wherein the permeation enhancer or combination of        permeation enhancers increases the flux of the therapeutic agent        or combination of therapeutic agents across a barrier; and    -   (c) a matrix forming agent or a combination of matrix forming        agents, wherein the matrix forming agent or combination of        matrix forming agents comprises a copolymer comprising        phosphoester monomers.

In another aspect, provided herein are compositions for treating an eardisease comprising:

-   -   (a) a therapeutic agent or a combination of therapeutic agents;    -   (b) a permeation enhancer or a combination of permeation        enhancers, wherein the permeation enhancer or combination of        permeation enhancers increases the flux of the therapeutic agent        or combination of therapeutic agents across the tympanic        membrane; and    -   (c) a matrix forming agent or a combination of matrix forming        agents, wherein the matrix forming agent or combination of        matrix forming agents comprises a copolymer comprising        phosphoester monomers.

In another aspect, provided herein are compositions comprising:

-   -   (a) a diagnostic agent or a combination of diagnostic agents;    -   (b) a permeation enhancer or a combination of permeation        enhancers, wherein the permeation enhancer or combination of        permeation enhancers increases the flux of the therapeutic agent        or combination of therapeutic agents across the tympanic        membrane; and    -   (c) a matrix forming agent or a combination of matrix forming        agents, wherein the matrix forming agent or combination of        matrix forming agents comprises a copolymer comprising        phosphoester monomers.

In another aspect, provided herein are compositions for treating aninfectious disease comprising:

-   -   (a) a therapeutic agent or a combination of therapeutic agents;    -   (b) a permeation enhancer or a combination of permeation        enhancers, wherein the permeation enhancer or combination of        permeation enhancers increases the flux of the therapeutic agent        or combination of therapeutic agents across a barrier; and    -   (c) a matrix forming agent or a combination of matrix forming        agents, wherein the matrix forming agent comprises a        polysaccharide derivative comprising cross-linkable functional        groups.

In another aspect, provided herein are compositions for treating an eardisease comprising:

-   -   (a) a therapeutic agent or a combination of therapeutic agents;    -   (b) a permeation enhancer or a combination of permeation        enhancers, wherein the permeation enhancer or combination of        permeation enhancers increases the flux of the therapeutic agent        or combination of therapeutic agents across the tympanic        membrane; and    -   (c) a matrix forming agent or a combination of matrix forming        agents, wherein the matrix forming agent comprises a        polysaccharide derivative comprising cross-linkable functional        groups.

The compositions provided herein typically include a permeation enhancer(e.g., a surfactant, terpene), a therapeutic agent (e.g., anantibiotic), and a matrix forming agent (e.g., a poloxamer derivative, apolyphosphoester containing polymer, a polysaccharide derivative). Thepermeation enhancer is an agent that alters the stratum corneum of thetympanic membrane to increase the flux of the therapeutic agent acrossthe tympanic membrane. The permeation enhancer facilitates delivery ofthe therapeutic agent into the middle and/or inner ear. Therapeuticagents include agents that have a therapeutic benefit in the ear. Incertain embodiments, the matrix forming agent is a liquid at ambientconditions, which once administered to a subject, gels (e.g., becomesmore viscous). In certain embodiments, the matrix forming agents gelsupon mixing of two components of the composition. In some embodiments,each component comprises a matrix forming agent (e.g., twopolysaccharide derivatives which undergo cross-linking upon mixing). Insome embodiments, one component comprises the matrix forming agent, andthe second component comprises an activator or catalyst which causesgelation when mixed with the matrix forming agent. In certainembodiments, the pharmaceutical composition does not substantiallyinterfere with the hearing of the subject.

Matrix Forming Agents

The matrix forming agent is a compound or mixture of compounds thatforms a gel after administration. In certain embodiments, the matrixforming agent forms a gel after administration into a subject's earcanal. The gel composition acts a reservoir containing the therapeuticagent and permeation enhancer, allowing for sustained release of thetherapeutic agent across a barrier (e.g., tympanic membrane). In certainembodiments, the gel maintains contact with the tympanic membrane. Insome embodiments, the gel maintains contact for between 0.5 and 1 hours,between 1 and 4 hours, between 1 and 8 hours, between 1 and 16 hours, orbetween 1 and 24 hours. In some embodiments, the gel maintains contactfor between 1 day and 3 days, between 1 and 7 days, or between 1 and 14days. In some embodiments, the gel allows flux of the therapeutic agentacross the tympanic membrane for between 0.5 and 1 hours, between 1 and4 hours, between 1 and 8 hours, between 1 and 16 hours, or between 1 and24 hours. In some embodiments, the gel maintains contact for between 1day and 3 days, between 1 and 7 days, or between 1 and 14 days. Such areservoir maintains contact with the tympanic membrane increasing thetime for the therapeutic agent to cross the tympanic membrane and bedelivered to the middle or inner ear. Such a reservoir maximizesexposure of the tympanic membrane to permeation enhancers and thetherapeutic agent, and facilitates sustained flux of the therapeuticagent into the middle and inner ear.

In various embodiments, the composition is a sustained releaseformulation. In various aspects, sustained release of either thepermeation enhancer and/or the therapeutic agent can be at a constantrate to deliver an effective amount of either the permeation enhancer ortherapeutic agent to the surface of the tympanic membrane, the middleear, or the inner ear. In various embodiments, the sustained releaseprovides a sufficient flux of therapeutic agent over about 1 day, about2 days, about 3 days, about 4 days, about 5 days, about 6 days, or about7 days. In various embodiments, the sustained release provides asufficient flux of therapeutic agent over a range of about 7 to about 10days. In various embodiments, the sustained release may be at a constantrate over a range of about 7 days to about 14 days. In variousembodiments, the sustained release provides a sufficient flux oftherapeutic agent over a range of about 14 to about 21 days. In variousembodiments, the sustained release provides a sufficient flux oftherapeutic agent over a range of about 21 to about 30 days. As usedherein, sufficient flux is the flux necessary for the therapeutic agentto be present in the middle ear in a therapeutically effective amount orprophylactically effective amount. In some embodiments, the sufficientflux is sufficient to provide an antibiotic agent in a concentrationequal or greater to the minimum inhibitory concentration of aninfectious microorganism. In some embodiments, the infectiousmicroorganism is H. influenza, S. pneumoniae, or M. catarrhalis.

In various aspects, the sustained release profile is obtained by theaddition of a matrix-forming agent to the composition. In variousembodiments, the composition may further comprise a matrix formingagent. In various embodiments, the matrix forming agents may undergo achange in viscosity, in situ, based on a phase change, a change insolubility, evaporation of a solvent, or mixing of components comprisingthe matrix forming agent. Such matrix forming agents gel, in situ afteradministration into a patient's ear canal to form a reservoir containingthe therapeutic agent and permeation enhancer, allowing sustainedrelease of the therapeutic agent. Such a reservoir maintains contactwith the tympanic membrane increasing the time for the therapeutic agentto permeate the tympanic membrane, and be delivered to the middle orinner ear. Such a reservoir maximizes exposure of the tympanic membraneto permeation enhancers and the therapeutic agent.

In certain embodiments, the matrix forming agent is a hydrogel, or formsa hydrogel upon administration. Matrix forming agents may include, butare not limited to, polyelectrolyte complexes, thermo-responsive gellingagents, pre-polymers, alginates, un-crosslinked polymers, and monomers,thermo-responsive gelling agents (e.g., poloxamer-polyphosphoestercopolymers), and polymers with cross-linkable functional groups. Incertain embodiments, the matrix forming agent is separated into a firstand second component which form a matrix or gel upon mixing. In someembodiments, a first matrix forming agent component is a first polymercomprising a first type of cross-linkable functional group, and a secondmatrix forming agent component is a second polymer comprising a secondtype of cross-linkable functional group, wherein the two types ofcross-linkable functional groups form cross-links between the twopolymers upon mixing of the first and second component. In someembodiments, a first matrix forming agent component comprises polymerswith cross-linkable functional groups, and a second matrix forming agentcomponent comprises an activator, wherein the cross-linkable functionalgroups form cross-links between the polymers upon mixing of the firstand second component. In some embodiments, the activator is an acid, abase, or a catalyst.

Matrix forming agents may further include biocompatible agents. Matrixforming agents may further include biodegradable agents. In certainembodiments the matrix forming agent is degraded and extruded from thebody of a patient within 3 days of application, within 7 days ofapplication, with 10 days of application, or within 14 days ofapplication. In various embodiments, the matrix forming agent has littleor no effect on hearing threshold when applied into a subject's earcanal. In various aspects, the matrix-forming agents may comprisebetween about 0 to about 40 percent of the composition. In variousembodiments, the matrix-forming agents may comprise between about 0 toabout 10 percent of the composition, comprise between about 10 to about20 percent of the composition, comprise between about 20 to about 30percent of the composition, comprise between about 30 to about 40percent of the composition, or comprise between about 40 to about 50percent of the composition.

The polymer may be a block copolymer. Exemplary polymer types suitablefor the block copolymer include, but are not limited to: polyethyleneoxide/polypropylene oxide based systems, poloxamers, poloxamer 407,poloxamer 188, poloxamines, methylcellulose, hydroxypropylmethylcellulose, ethyl(hydroxyethyl) cellulose, xyloglucan, cellulose,acetate phthalate, latex, poly(acrylic acid), thermoresponsivepolysaccharides (including cellulose derivatives, chitosan, dextran andgellan gum). In some embodiments, the matrix forming agent comprises apolyethylene/polypropylene copolymer or polyethylene/polypropylene blockcopolymer. In some embodiments, the matrix forming agent comprises apoloxamer. In some embodiments, the matrix forming agent comprisespoloxamer 407, poloxamer 188, poloxalene, poloxamer 124, poloxamer 237,or poloxamer 338.

Exemplary poloxamers include, but are not limited to: poloxamer 407,poloxamer 188, poloxalene, poloxamer 124, poloxamer 237, or poloxamer338, Pluronic® 10R5, Pluronic® 17R2, Pluronic® 17R4, Pluronic® 25R2,Pluronic® 25R4, Pluronic® 31R1, Pluronic® F 108 Cast Solid Surfactant,Pluronic® F 108 NF, Pluronic® F 108 Pastille, Pluronic® F 108NF PrillPoloxamer 338, Pluronic® F 127 NF, Pluronic® F 127 NF 500 BHT Prill,Pluronic® F 127 NF Prill Poloxamer 407, Pluronic® F 38, Pluronic® F 38Pastille, Pluronic® F 68, Pluronic® F 68 LF Pastille, Pluronic® F 68 NF,Pluronic® F 68 NF Prill Poloxamer 188, Pluronic® F 68 Pastille,Pluronic® F 77, Pluronic® F 77 Micropastille, Pluronic® F 87, Pluronic®F 87 NF, Pluronic® F 87 NF Prill Poloxamer 237, Pluronic® F 88,Pluronic® F 88 Pastille, Pluronic® FT L 61, Pluronic® L 10, Pluronic® L101, Pluronic® L 121, Pluronic® L 31, Pluronic® L 35, Pluronic® L 43,Pluronic® L 61, Pluronic® L 62, Pluronic® L 62 LF, Pluronic® L 62D,Pluronic® L 64, Pluronic® L 81, Pluronic® L 92, Pluronic® L44 NF INHsurfactant Poloxamer 124, Pluronic® N 3, Pluronic® P 103, Pluronic® P104, Pluronic® P 105, Pluronic® P 123 Surfactant, Pluronic® P 65,Pluronic® P 84, Pluronic® P 85, Synperonic® PE/F 108, Synperonic®PE/P105, Synperonic® PE/P84, Synperonic®, Synperonic® PE/L31,Synperonic® PE/L61, Synperonic® PE/L101, Synperonic® PE/L121,Synperonic® PE/L42, Synperonic® PE/L62, Synperonic® PE/L92, Synperonic®PE/L44, Synperonic® PE/L64, Synperonic® PE/P84, Synperonic® PE/P75,Synperonic® PE/P103, Synperonic® PE/F87, Synperonic® PE/F127,Synperonic® PE/F38, Synperonic® PE/F68, Kolliphor® P 188, Kolliphor® P407, Kolliphor® P 188 micro, Kolliphor® P 407 micro, Kolliphor® P237,Kolliphor® P 338, Kolliphor® EL, Kolliphor® HS 15, Kolliphor® PS 80,Kolliphor® PS 60, Kolliphor® RH 40, Kolliphor® TPG S, Kolliphor® CS L,Kolliphor® CS A, Kolliphor® CS S, Kolliphor® CS B, Kolliphor® CS 20, andKolliphor® CS 12. In some embodiments, the matrix forming agentcomprises any of the foregoing poloxamers, a derivative thereof, or ablock copolymer thereof.

In various embodiments, of the present inventions the polyelectrolytecomplex may include, but is not limited to a, chitosan-chondroitinsulfate complex, gelatin, carboxymethycellulose, glycosaminoglycans andpoly (vinyl alcohol). In various aspects, the relative ratios ofchitosan to chondroitin sulfate may be between about 1:0.09 to about1:1.4. In certain embodiments, the polyelectrolyte complex is achitosan-chondroitin sulfate complex.

In certain embodiments, the percent weight of matrix forming agent inthe composition is between about 1% to about 10%, between about 10% toabout 20%, between about 20% to about 30%, between about 30% to about40%, between about 40% to about 50%, or between about 50% to about 90%.In some embodiments, the percent weight of matrix forming agent in thecomposition is between 1% to about 10%. In some embodiments, the percentweight of matrix forming agent in the composition is between about 10%to about 20%. In some embodiments, the percent weight of matrix formingagent in the composition is between 20% to about 30%.

Polymers Comprising Polyphosphoester Monomers or Blocks

In certain embodiments, the matrix forming comprises a copolymer withphosphoester monomers. The copolymer comprises at least one phosphoestermonomer and at least one non-phosphoester monomer. In some embodiments,the copolymer comprises a polyphosphoester block. In some embodiments,the copolymer comprises a polyphosphoester block and anon-polyphosphoester block. In some embodiments, the block copolymercomprises a polyphosphoester block, and another blocks selected from thegroup consisting of polyethylene oxide, polypropylene oxide, poloxamers,poloxamer 407, poloxamer 188, poloxamines, methylcellulose,hydroxypropyl methylcellulose, ethyl(hydroxyethyl) cellulose,xyloglucan, acetates, phthalates, latex, poly(acrylic acid),N-isopropylacrylamides, cellulose, chitosan, dextran, hyaluronic acid,and derivatives thereof. In some embodiments, the polymer comprisesthermoresponsive polysaccharides (e.g., cellulose derivatives, chitosan,dextran and gellan gum). In some embodiments, the block copolymercomprises polyphosphoester blocks, and comprises apolyethylene/polypropylene copolymer or polyethylene/polypropylene blockcopolymer. In some embodiments, the block copolymer comprisespolyphosphoester blocks, and comprises a poloxamer. In some embodiments,the composition has a high degree of hydrophobicity. In someembodiments, the block copolymer has a high degree of hydrophobicity. Insome embodiments, the composition is optically transparent. In someembodiments, the block copolymer comprises polyphosphoester blocks, andcomprises poloxamer 407. In some embodiments, each block containsbetween 1 and 200 monomers.

In certain embodiments, the polyphosphoester copolymer orpolyphosphoester blocks comprise monomers of Formula (M):

wherein for each monomer, Y is independently —R¹ or -L²R², wherein:

-   -   each occurrence of R¹ is independently hydrogen, optionally        substituted alkyl, optionally substituted alkenyl, optionally        substituted alkynyl, optionally substituted aryl, or optionally        substituted heteroaryl;    -   each occurrence of L² is independently a bond, optionally        substituted alkylene, optionally substituted alkenylene,        optionally substituted alkynylene, optionally substituted        heteroalkylene, optionally substituted heteroalkenylene, or        optionally substituted heteroalkynylene;    -   each occurrence of R² is independently optionally substituted        acyl, optionally substituted carbocyclyl, optionally substituted        heterocyclyl, optionally substituted aryl, optionally        substituted heteroaryl, —OR^(b), —N(R^(b))₂, or an oxygen        protecting group; and    -   each occurrence of R^(b) is independently optionally substituted        alkyl, optionally substituted alkenyl, optionally substituted        alkynyl, optionally substituted carbocyclyl, optionally        substituted heterocyclyl, optionally substituted aryl,        optionally substituted heteroaryl, optionally substituted acyl,        an oxygen protecting group, or a nitrogen protecting group, or        two R^(b) taken together with the nitrogen to which they are        attached form an optionally substituted heterocyclic or        optionally substituted heteroaryl ring.

In certain embodiments, the polyphosphoester copolymer orpolyphosphoester blocks comprise monomers of Formula (M-i):

wherein R¹ is as defined herein.

In certain embodiments, the polyphosphoester copolymer orpolyphosphoester blocks comprise monomers of Formula (M-ii):

wherein L² and R² are as defined herein.

In certain embodiments, the matrix forming agent comprisespolyphosphoester blocks comprising monomers of Formula (M-i), andpolyphosphoester blocks comprising monomers of formula (M-ii). In someembodiments, the matrix forming agent comprises polyphosphoester blockscomprising monomers of both Formulae (M-i) and (M-ii)

In certain embodiments, the matrix forming agent is a polymer of Formula(I):

wherein:

-   -   each occurrence of Y is independently —R¹ or -L²R²;    -   each occurrence of R¹ is independently hydrogen, optionally        substituted alkyl, optionally substituted alkenyl, optionally        substituted alkynyl, optionally substituted aryl, or optionally        substituted heteroaryl;    -   each occurrence of L² is independently a bond, optionally        substituted alkylene, optionally substituted alkenylene,        optionally substituted alkynylene, optionally substituted        heteroalkylene, optionally substituted heteroalkenylene, or        optionally substituted heteroalkynylene;    -   each occurrence of R² is independently optionally substituted        acyl, optionally substituted carbocyclyl, optionally substituted        heterocyclyl, optionally substituted aryl, optionally        substituted heteroaryl, —OR^(b), —N(R^(b))₂, or an oxygen        protecting group;    -   each occurrence of R³ is independently optionally substituted        alkyl, optionally substituted alkenyl, optionally substituted        alkynyl, optionally substituted aryl, optionally substituted        heteryaryl, optionally substituted acyl, —OR^(b), or —N(R^(b))₂;    -   each occurrence of R^(b) is independently optionally substituted        alkyl, optionally substituted alkenyl, optionally substituted        alkynyl, optionally substituted carbocyclyl, optionally        substituted heterocyclyl, optionally substituted aryl,        optionally substituted heteroaryl, optionally substituted acyl,        an oxygen protecting group, or a nitrogen protecting group, or        two R^(b) taken together with the nitrogen to which they are        attached form an optionally substituted heterocyclic or        optionally substituted heteroaryl ring;    -   each of G¹ and G² is independently hydrogen, optionally        substituted alkyl, optionally substituted aryl, or optionally        substituted heteroaryl, optionally substituted acyl, optionally        substituted phosphate, or an oxygen protecting group; and    -   each of p, q, r, s, and t is independently an integer between 0        and 200, inclusive, wherein the sum of p and t is at least 1,        and the sum of q, r, and s is at least 1.

Phosphoester monomers and polyphosphoester blocks described herein maycomprise group Y. In certain embodiments, each Y in a copolymer is thesame substituent. In certain embodiments, each Y in a copolymer is oneof two specific substituents. In certain embodiments, each Y in acopolymer is one of three specific substituents. In certain embodiments,each Y in a copolymer is one of four specific substituents. In certainembodiments, each Y in a copolymer is one of five specific substituents.In certain embodiments, each Y in a copolymer is one of six specificsubstituents. In certain embodiments, each Y in a copolymer is one ofseven or more specific substituents. In some embodiments, Y is —R¹. Insome embodiments, Y is -L²R².

In certain embodiments, p is 0. In certain embodiments, p is an integerbetween 1 and 100, inclusive. In some embodiments, p is an integerbetween 10 and 100, inclusive. In some embodiments, p is an integerbetween 10 and 50, inclusive. In some embodiments, p is an integerbetween 10 and 25, inclusive. In some embodiments, p is an integerbetween 1 and 10, inclusive.

In certain embodiments, t is 0. In certain embodiments, t is an integerbetween 1 and 100, inclusive. In some embodiments, t is an integerbetween 10 and 100, inclusive. In some embodiments, t is an integerbetween 10 and 50, inclusive. In some embodiments, t is an integerbetween 10 and 25, inclusive. In some embodiments, t is an integerbetween 1 and 10, inclusive.

In certain embodiments, q is 0. In certain embodiments, q is an integerbetween 1 and 100, inclusive. In some embodiments, q is an integerbetween 10 and 100, inclusive. In some embodiments, q is an integerbetween 10 and 50, inclusive. In some embodiments, q is an integerbetween 10 and 25, inclusive. In some embodiments, q is an integerbetween 1 and 10, inclusive.

In certain embodiments, r is 0. In certain embodiments, r is an integerbetween 1 and 100, inclusive. In some embodiments, r is an integerbetween 10 and 100, inclusive. In some embodiments, r is an integerbetween 10 and 50, inclusive. In some embodiments, r is an integerbetween 10 and 25, inclusive. In some embodiments, r is an integerbetween 1 and 10, inclusive.

In certain embodiments, s is 0. In certain embodiments, s is an integerbetween 1 and 100. In some embodiments, s is an integer between 10 and100, inclusive. In some embodiments, s is an integer between 10 and 50,inclusive. In some embodiments, s is an integer between 10 and 25,inclusive. In some embodiments, s is an integer between 1 and 10,inclusive. In certain embodiments, both q and s are 0. In certainembodiments, exactly one of q and s is 0.

G¹ and G² are terminal groups of the polymer. As generally definedherein G¹ and G² are hydrogen, optionally substituted alkyl, optionallysubstituted aryl, optionally substituted heteroaryl, optionallysubstituted acyl, optionally substituted phosphate, or an oxygenprotecting group. In some embodiments, G¹ and G² are the same. In someembodiments, G¹ and G² are both hydrogen. In some embodiments, G¹ and G²are different.

In certain embodiments, G¹ is hydrogen. In certain embodiments, G¹ isoptionally substituted alkyl. In certain embodiments, G¹ is optionallysubstituted acyl. In certain embodiments, G¹ is optionally substitutedoptionally substituted phosphate (e.g., —P(═O)(OH)₂, —P(═O)(O-alkyl)₂,—P(═O)(OH)(O-alkyl), —P(═O)(OH)(O—Y), —P(═O)(O-alkyl)(O—Y)). In certainembodiments, G¹ is an oxygen protecting group (e.g., silyl, TBDPS,TBDMS, TIPS, TES, TMS, MOM, THP, t-Bu, Bn, allyl, acetyl, pivaloyl,benzoyl). In certain embodiments, G¹ is hydrogen. In certainembodiments, G¹ is optionally substituted alkyl. In certain embodiments,G¹ is optionally substituted acyl. In certain embodiments, G¹ isoptionally substituted optionally substituted phosphate (e.g.,—P(═O)(OH)₂, —P(═O)(O-alkyl)₂, —P(═O)(OH)(O-alkyl), —P(═O)(OH)(O—Y),—P(═O)(O-alkyl)(O—Y)). In certain embodiments, G¹ is an oxygenprotecting group (e.g., silyl, TBDPS, TBDMS, TIPS, TES, TMS, MOM, THP,t-Bu, Bn, allyl, acetyl, pivaloyl, benzoyl). In certain embodiments, G¹is optionally substituted aryl, e.g., optionally substituted phenyl. Incertain embodiments, G¹ is unsubstituted aryl, e.g., unsubstitutedphenyl. In certain embodiments, G¹ is optionally substituted heteroaryl,e.g., optionally substituted 5-6 membered heteroaryl, or optionallysubstituted 9-10 membered bicyclic heteroaryl. In certain embodiments,G¹ is unsubstituted heteroaryl, e.g., unsubstituted 5-6 memberedheteroaryl, or unsubstituted 9-10 membered bicyclic heteroaryl.

In certain embodiments, G² is hydrogen. In certain embodiments, G² isoptionally substituted alkyl. In certain embodiments, G² is optionallysubstituted acyl. In certain embodiments, G² is optionally substitutedoptionally substituted phosphate (e.g., —P(═O)(OH)₂, —P(═O)(O-alkyl)₂,—P(═O)(OH)(O-alkyl), —P(═O)(OH)(O—Y), —P(═O)(O-alkyl)(O—Y)). In certainembodiments, G² is an oxygen protecting group (e.g., silyl, TBDPS,TBDMS, TIPS, TES, TMS, MOM, THP, t-Bu, Bn, allyl, acetyl, pivaloyl,benzoyl). In certain embodiments, G² is hydrogen. In certainembodiments, G² is optionally substituted alkyl. In certain embodiments,G² is optionally substituted acyl. In certain embodiments, G² isoptionally substituted optionally substituted phosphate (e.g.,—P(═O)(OH)₂, —P(═O)(O-alkyl)₂, —P(═O)(OH)(O-alkyl), —P(═O)(OH)(O—Y),—P(═O)(O-alkyl)(O—Y)). In certain embodiments, G² is an oxygenprotecting group (e.g., silyl, TBDPS, TBDMS, TIPS, TES, TMS, MOM, THP,t-Bu, Bn, allyl, acetyl, pivaloyl, benzoyl). In certain embodiments, G²is optionally substituted aryl, e.g., optionally substituted phenyl. Incertain embodiments, G² is unsubstituted aryl, e.g., unsubstitutedphenyl. In certain embodiments, G² is optionally substituted heteroaryl,e.g., optionally substituted 5-6 membered heteroaryl, or optionallysubstituted 9-10 membered bicyclic heteroaryl. In certain embodiments,G² is unsubstituted heteroaryl, e.g., unsubstituted 5-6 memberedheteroaryl, or unsubstituted 9-10 membered bicyclic heteroaryl.

In general, increasing PPE hydrophobicity decreases the gelationtemperature, and accelerates gelation kinetics. Hydrophobicity of the PEmonomer may be tuned by selection of pendent group Y (See, e.g., FIG.7). In certain embodiments, greater numbers of hydrophobic domains willmake the polymer more resistant to the effect of the CPEs on micelleformation than P407. The fact that P407-PPE may form gels not only bymicelle formation but also by the hydrophobic PPE domains forming across-linked network [46] may also have a similar effect, and create anadditional means of tuning phase transition behavior. It is noted thatexcessively hydrophobic PPE blocks may induce polymer aggregation inwater, which could be undesirable for administration.

The bioadhesion of P407 can be enhanced by the conjugation ofpoly(acrylic acid) (PAA), or other reactive groups. In certainembodiments, PE monomers are functionalized with carbonyl and/or withacrylate groups (See, e.g., FIG. 7). In certain embodiments, PE monomerswith hydrophobic groups and PE monomers with bioadhesion groups are bothincorporated in the matrix forming agent. In some embodiments, thehydrophobic groups and bioadhesion groups are incorporated in the samepolymer. In some embodiments, the hydrophobic groups and bioadhesiongroups are incorporated in separate polymers.

R¹

Phosphoester monomers and polyphosphoester blocks described herein maycomprise R¹. In certain embodiments, each R¹ is the same substituent. Incertain embodiments, each R¹ in a polymer is one of two specificsubstituents. In certain embodiments, each R¹ in a polymer is one ofthree specific substituents. In certain embodiments, each R¹ in apolymer is one of four specific substituents. In certain embodiments,each R¹ in a polymer is one of five specific substituents. In certainembodiments, each R¹ in a polymer is one of six specific substituents.In certain embodiments, each R¹ in a polymer is one of seven or morespecific substituents.

As generally described herein each occurrence of R¹ is independentlyhydrogen, optionally substituted alkyl, optionally substituted alkenyl,or optionally substituted alkynyl. In certain embodiments, eachoccurrence of R¹ is independently hydrogen or optionally substitutedalkyl. In certain embodiments, each occurrence of R¹ is independentlyhydrogen or unsubstituted alkyl.

In certain embodiments, R¹ is hydrogen. In certain embodiments, R^(A1)is a non-hydrogen group.

In certain embodiments, R¹ is optionally substituted alkyl, e.g.,optionally substituted C₁₋₆ alkyl, optionally substituted C₁₋₂ alkyl,optionally substituted C₂₋₃ alkyl, optionally substituted C₃₋₄ alkyl,optionally substituted C₄₋₅ alkyl, or optionally substituted C₅₋₆ alkyl.In certain embodiments, R¹ is unsubstituted alkyl, e.g., unsubstitutedC₁₋₆ alkyl, unsubstituted C₁₋₂ alkyl, unsubstituted C₂₋₃ alkyl,unsubstituted C₃₋₄ alkyl, unsubstituted C₄₋₅ alkyl, or unsubstitutedC₅₋₆ alkyl. In certain embodiments, R¹ is unsubstituted C₁₋₂₀ alkyl. Incertain embodiments, R¹ is unsubstituted C₁₋₁₂ alkyl. In In certainembodiments, R¹ is methyl. In certain embodiments, R¹ is ethyl, propyl,or butyl. In certain embodiments, R¹ is haloalkyl, e.g., —CHF₂, —CHCl₂,—CH₂CHF₂, —CH₂CHCl₂. In certain embodiments, R¹ is perhaloalkyl, e.g.,—CF₃, —CF₂CF₃, —CCl₃. In certain embodiments, R¹ is hydroxyalkyl, e.g.,—CH₂OH, —CH₂CH₂OH, —CH₂OR^(b), —CH₂CH₂OR^(b). In certain embodiments, R¹is aminoalkyl, e.g., —CH₂NH₂, —CH₂CH₂NH₂, —CH₂NMe₂, —CH₂CH₂NMe₂,—CH₂N(R^(b))₂, —CH₂CH₂N(R^(b))₂.

In certain embodiments, R¹ is optionally substituted alkenyl, e.g.,optionally substituted C₂₋₆ alkenyl. In certain embodiments, R¹ isunsubstituted alkenyl, e.g., unsubstituted C₂₋₆ alkenyl. In certainembodiments, R¹ is vinyl, allyl, or prenyl. In certain embodiments, R¹is optionally substituted alkynyl, e.g., optionally substituted C₂₋₆alkynyl. In certain embodiments, R¹ is unsubstituted alkynyl, e.g.,unsubstituted C₂₋₆ alkynyl.

In certain embodiments, R¹ is unsubstituted, unbranched C₁₋₂₀ alkyl. Incertain embodiments, R¹ is unsubstituted, branched C₁₋₂₀ alkyl. Incertain embodiments, R¹ is of formula:

wherein each occurrence of n is independently an integer between 0 and20, inclusive.

In certain embodiments, R¹ is of formula:

wherein each occurrence of n is independently an integer between 0 and20, inclusive.

In certain embodiments, R¹ is of formula:

In certain embodiments, R¹ is optionally substituted aryl, e.g.,optionally substituted phenyl. In certain embodiments, R¹ isunsubstituted aryl, e.g., unsubstituted phenyl. In certain embodiments,R¹ is optionally substituted heteroaryl, e.g., optionally substituted5-6 membered heteroaryl, or optionally substituted 9-10 memberedbicyclic heteroaryl. In certain embodiments, R¹ is unsubstitutedheteroaryl, e.g., unsubstituted 5-6 membered heteroaryl, orunsubstituted 9-10 membered bicyclic heteroaryl.

L² and R²

Phosphoester monomers and polyphosphoester blocks described herein maycomprise -L²R². In certain embodiments, each -L²R² in a copolymer is thesame substituent. In certain embodiments, each -L²R² in a copolymer isone of two specific substituents. In certain embodiments, each -L²R² ina copolymer is one of three specific substituents. In certainembodiments, each -L²R² in a copolymer is one of four specificsubstituents. In certain embodiments, each -L²R² in a copolymer is oneof five specific substituents. In certain embodiments, each -L²R² in acopolymer is one of six specific substituents. In certain embodiments,each -L²R² in a copolymer is one of seven or more specific substituents.

As generally described herein, each occurrence of L² is optionallysubstituted alkylene, optionally substituted alkenylene, optionallysubstituted alkynylene, optionally substituted heteroalkylene,optionally substituted heteroalkenylene, or optionally substitutedheteroalkynylene. In certain embodiments, each L² is independentlyoptionally substituted alkylene. In certain embodiments, each L² isindependently optionally substituted alkylene, and each occurrence of R²is independently optionally substituted acyl. In certain embodiments,each L² is independently unsubstituted alkylene. In certain embodiments,each L² is independently unsubstituted alkylene, and each occurrence ofR² is independently optionally substituted acyl.

In certain embodiments, L² is optionally substituted alkylene, e.g.,optionally substituted C₁₋₆ alkylene, optionally substituted C₁₋₂alkylene, optionally substituted C₂₋₃ alkylene, optionally substitutedC₃₋₄ alkylene, optionally substituted C₄₋₅ alkylene, or optionallysubstituted C₅₋₆ alkylene. In certain embodiments, L² is unsubstitutedalkylene, e.g., unsubstituted C₁₋₆ alkylene, unsubstituted C₁₋₂alkylene, unsubstituted C₂₋₃ alkylene, unsubstituted C₃₋₄ alkylene,unsubstituted C₄₋₅ alkylene, or unsubstituted C₅₋₆ alkylene. In certainembodiments, L² is methylene. In certain embodiments, L² is ethylene,propylene, butylene, pentylene, or hexylene.

In certain embodiments, L² is optionally substituted alkenylene, e.g.,optionally substituted C₂₋₆ alkenylene. In certain embodiments, L² isunsubstituted alkenylene, e.g., unsubstituted C₂₋₆ alkenylene. Incertain embodiments, L² is vinylene, allylene, or prenylene. In certainembodiments, L² is optionally substituted alkynylene, e.g., optionallysubstituted C₂₋₆ alkynylene. In certain embodiments, L² is unsubstitutedalkynylene, e.g., unsubstituted C₂₋₆ alkynylene.

In certain embodiments, L² is optionally substituted heteroalkylene,e.g., optionally substituted C₁₋₆ heteroalkylene. In some embodiments,L² is unsubstituted heteroalkylene, wherein the heteroalkylene containsone oxygen atom. In some embodiments, L² is unsubstitutedheteroalkylene, wherein the heteroalkylene contains one nitrogen atom.In certain embodiments, L² is optionally substituted heteroalkenylene,e.g., optionally substituted C₁₋₆ heteroalkenylene. In some embodiments,L² is unsubstituted heteroalkenylene, wherein the heteroalkenylenecontains one oxygen atom. In some embodiments, L² is unsubstitutedheteroalkenylene, wherein the heteroalkylene contains one nitrogen atom.

As generally described herein each occurrence of R² is independentlyoptionally substituted acyl, optionally substituted carbocyclyl,optionally substituted heterocyclyl, optionally substituted aryl,optionally substituted heteroaryl, —OR^(b), —N(R^(b))₂, or an oxygenprotecting group. In certain embodiments, each R² is independentlyoptionally substituted acyl. In certain embodiments, each R² isindependently optionally substituted acyl, and each occurrence of L² isindependently optionally substituted alkylene.

In certain embodiments, R² is optionally substituted carbocyclyl, e.g.,optionally substituted C₃₋₆ carbocyclyl, optionally substituted C₃₋₄carbocyclyl, optionally substituted C₄₋₅ carbocyclyl, or optionallysubstituted C₅₋₆ carbocyclyl. In certain embodiments, R² isunsubstituted carbocyclyl, e.g., unsubstituted C₃₋₆ carbocyclyl. In someembodiments, R² is cyclopropyl, cyclobutyl, cyclopentyl, or cyclohexyl.In certain embodiments, R² is optionally substituted heterocyclyl, e.g.,optionally substituted 3-6 membered heterocyclyl, optionally substituted3-4 membered heterocyclyl, optionally substituted 4-5 memberedheterocyclyl, or optionally substituted 5-6 membered heterocyclyl. Incertain embodiments, R² is unsubstituted heterocyclyl, e.g.,unsubstituted 3-6 membered heterocyclyl, unsubstituted 3-4 memberedheterocyclyl, unsubstituted 4-5 membered heterocyclyl, or unsubstituted5-6 membered heterocyclyl.

In certain embodiments, R² is optionally substituted aryl, e.g.,optionally substituted phenyl. In certain embodiments, R² isunsubstituted aryl, e.g., unsubstituted phenyl. In certain embodiments,R² is optionally substituted heteroaryl, e.g., optionally substituted5-6 membered heteroaryl, or optionally substituted 9-10 memberedbicyclic heteroaryl. In certain embodiments, R² is unsubstitutedheteroaryl, e.g., unsubstituted 5-6 membered heteroaryl, orunsubstituted 9-10 membered bicyclic heteroaryl.

In certain embodiments, R² is optionally substituted acyl, e.g., —CHO,—CO₂H, or —C(═O)NH₂. In certain embodiments, R² is optionallysubstituted carbonyl. In certain embodiments, R² is —C(═O)R^(b),—C(═O)OR^(b), —C(═O)NH(R^(b)), or —C(═O)N(R^(b))₂. In certainembodiments, R² is —C(═O)R^(b), and R^(b) is optionally substitutedalkyl, e.g., —C(═O)Me. In certain embodiments, R² is —C(═O)R^(b), andR^(b) is optionally substituted alkenyl. In certain embodiments, R² is—C(═O)R^(b), and R^(b) is optionally substituted carbocyclyl,heterocyclyl, aryl, or heteroaryl. In certain embodiments, R² is—C(═O)OR^(b), and R^(b) is optionally substituted alkyl. In certainembodiments, R² is —C(═O)OR^(b), and R^(b) is optionally substitutedalkenyl. In certain embodiments, R² is —C(═O)OR^(b), and R^(b) isoptionally substituted carbocyclyl, heterocyclyl, aryl, or heteroaryl.In certain embodiments, R² is —C(═O)N(R^(b))₂, and at least one R^(b) isoptionally substituted alkyl. In certain embodiments, R² is—C(═O)NHR^(b), and R^(b) is optionally substituted alkyl. In certainembodiments, R² is —C(═O)NHR^(b), and R^(b) is optionally substitutedalkenyl. In certain embodiments, R² is —C(═O)NHR^(b), and R^(b) isoptionally substituted carbocyclyl, heterocyclyl, aryl, or heteroaryl.In certain embodiments, R² is optionally substituted vinylcarbonyl(e.g., —C(═O)CH═CH₂, —C(═O)CMe=CH₂). In certain embodiments, R² is anoxygen protecting group (e.g., silyl, TBDPS, TBDMS, TIPS, TES, TMS, MOM,THP, t-Bu, Bn, allyl, acetyl, pivaloyl, benzoyl)

In certain embodiments, R² is —OR^(b), e.g., —OH. In certainembodiments, R² is —OR^(b), and R^(b) is optionally substituted alkyl.In certain embodiments, R² is —OR^(b), and R^(b) is unsubstituted C₁₋₆alkyl. In certain embodiments, R² is —OR^(b), and R^(b) is optionallysubstituted alkenyl. In certain embodiments, R² is —OR^(b), and R^(b) isoptionally substituted carbocyclyl, optionally substituted heterocyclyl,optionally substituted aryl optionally substituted heteroaryl. Incertain embodiments, R² is —OR^(b), and R^(b) is unsubstitutedcarbocyclyl, unsubstituted heterocyclyl, unsubstituted arylunsubstituted heteroaryl. In certain embodiments, R² is —OR^(b), andR^(b) is optionally substituted acyl, e.g., R² is —OC(═O)R^(b),—OC(═O)OR^(b), or —OC(═O)N(R^(b))₂. In certain embodiments, R² is—OR^(b), and R^(b) is an oxygen protecting group (e.g., silyl, TBDPS,TBDMS, TIPS, TES, TMS, MOM, THP, t-Bu, Bn, allyl, acetyl, pivaloyl,benzoyl).

In certain embodiments, R² is —N(R^(b))₂, e.g., —NH₂, —NHR^(b). Incertain embodiments, R² is —NH(R^(b)), and R^(b) is optionallysubstituted alkyl. In certain embodiments, R² is —N(R^(b))₂, and atleast one R^(b) is optionally substituted alkyl. In certain embodiments,R² is —NH(R^(b)), and R^(b) is unsubstituted alkyl. In certainembodiments, R² is —N(R^(b))₂, and at least one R^(b) is unsubstitutedalkyl. In certain embodiments, R² is —NHR^(b), and R^(b) is optionallysubstituted carbocyclyl, optionally substituted heterocyclyl, optionallysubstituted aryl, or optionally substituted heteroaryl. In certainembodiments, R² is —NHR^(b), and R^(b) is unsubstituted carbocyclyl,unsubstituted heterocyclyl, unsubstituted aryl, or unsubstitutedheteroaryl. In certain embodiments, R² is —NHR^(b), and R^(b) isoptionally substituted acyl, e.g., R² is —NHC(═O)R^(b), —NHC(═O)OR^(b),or —NHC(═O)NHR^(b). In certain embodiments, R² is —N(R^(b))₂, and atleast one R^(b) is a nitrogen protecting group (e.g., Bn, Boc, Cbz,Fmoc, trifluoroacetyl, triphenylmethyl, acetyl, Ts). In certainembodiments, R² is —N(R^(b))₂, and both R^(b) are joined to form anoptionally substituted heterocyclic or optionally substituted heteroarylring. In certain embodiments, R² is —N(R^(b))₂, and both R^(b) arejoined to form an unsubstituted heterocyclic or unsubstituted heteroarylring.

In certain embodiments, -L²R² is of formula:

wherein n is an integer between 0 and 20, inclusive.

In certain embodiments, -L²R² is of formula:

wherein n is an integer between 0 and 20, inclusive.

In certain embodiments, -L²R² is of formula:

In certain embodiments, -L²R² is of formula:

R³

Polymers of Formula (I) comprise R³. In certain embodiments, each R³ isthe same substituent. In certain embodiments, each R³ in a polymer isone of two specific substituents. In certain embodiments, each R³ in apolymer is one of three specific substituents. In certain embodiments,each R³ in a polymer is one of four specific substituents. In certainembodiments, each R³ in a polymer is one of five specific substituents.In certain embodiments, each R³ in a polymer is one of six specificsubstituents. In certain embodiments, each R³ in a polymer is one ofseven or more specific substituents.

As generally described herein each occurrence of R³ is independentlyoptionally substituted alkyl, optionally substituted alkenyl, optionallysubstituted alkynyl, optionally substituted aryl, optionally substitutedheteryaryl, optionally substituted acyl, —OR^(b), or —N(R^(b))₂. Incertain embodiments, each occurrence of R³ is independentlyunsubstituted alkyl.

In certain embodiments, R³ is optionally substituted alkyl, e.g.,optionally substituted Ci-6 alkyl, optionally substituted C₁₋₂ alkyl,optionally substituted C₂₋₃ alkyl, optionally substituted C₃₋₄ alkyl,optionally substituted C₄₋₅ alkyl, or optionally substituted C₅₋₆ alkyl.In certain embodiments, R³ is unsubstituted alkyl, e.g., unsubstitutedC₁₋₆ alkyl, unsubstituted C₁₋₂ alkyl, unsubstituted C₂₋₃ alkyl,unsubstituted C₃₋₄ alkyl, unsubstituted C₄₋₅ alkyl, or unsubstitutedC₅₋₆ alkyl. In certain embodiments, R³ is unsubstituted C₁₋₂₀ alkyl. Incertain embodiments, R³ is unsubstituted C₁₋₁₂ alkyl. In certainembodiments, R³ is methyl. In certain embodiments, R³ is ethyl, propyl,or butyl. In certain embodiments, R³ is haloalkyl, e.g., —CHF₂, —CHCl₂,—CH₂CHF₂, —CH₂CHCl₂. In certain embodiments, R³ is perhaloalkyl, e.g.,—CF₃, —CF₂CF₃, —CCl₃. In certain embodiments, R³ is hydroxyalkyl, e.g.,—CH₂OH, —CH₂CH₂OH, —CH₂OR^(b), —CH₂CH₂OR^(b). In certain embodiments, R³is aminoalkyl, e.g., —CH₂NH₂, —CH₂CH₂NH₂, —CH₂NMe₂, —CH₂CH₂NMe₂,—CH₂N(R^(b))₂, —CH₂CH₂N(R^(b))₂.

In certain embodiments, R³ is optionally substituted alkenyl, e.g.,optionally substituted C₂₋₆ alkenyl. In certain embodiments, R³ isunsubstituted alkenyl, e.g., unsubstituted C₂₋₆ alkenyl. In certainembodiments, R³ is vinyl, allyl, or prenyl. In certain embodiments, R³is optionally substituted alkynyl, e.g., optionally substituted C₂₋₆alkynyl. In certain embodiments, R³ is unsubstituted alkynyl, e.g.,unsubstituted C₂₋₆ alkynyl.

In certain embodiments, R³ is optionally substituted aryl, e.g.,optionally substituted phenyl. In certain embodiments, R³ isunsubstituted aryl, e.g., unsubstituted phenyl. In certain embodiments,R³ is optionally substituted heteroaryl, e.g., optionally substituted5-6 membered heteroaryl, or optionally substituted 9-10 memberedbicyclic heteroaryl. In certain embodiments, R³ is unsubstitutedheteroaryl, e.g., unsubstituted 5-6 membered heteroaryl, orunsubstituted 9-10 membered bicyclic heteroaryl.

In certain embodiments, R³ is optionally substituted acyl, e.g., —CHO,—CO₂H, or —C(═O)NH₂. In certain embodiments, R³ is optionallysubstituted carbonyl. In certain embodiments, R³ is —C(═O)R^(b),—C(═O)OR^(b), —C(═O)NH(R^(b)), or —C(═O)N(R^(b))₂. In certainembodiments, R³ is —C(═O)R^(b), and R^(b) is optionally substitutedalkyl, e.g., —C(═O)Me. In certain embodiments, R³ is —C(═O)R^(b), andR^(b) is optionally substituted alkenyl. In certain embodiments, R³ is—C(═O)R^(b), and R^(b) is optionally substituted carbocyclyl,heterocyclyl, aryl, or heteroaryl. In certain embodiments, R³ is—C(═O)OR^(b), and R^(b) is optionally substituted alkyl. In certainembodiments, R³ is —C(═O)OR^(b), and R^(b) is optionally substitutedalkenyl. In certain embodiments, R³ is —C(═O)OR^(b), and R^(b) isoptionally substituted carbocyclyl, heterocyclyl, aryl, or heteroaryl.In certain embodiments, R³ is —C(═O)N(R^(b))₂, and at least one R^(b) isoptionally substituted alkyl. In certain embodiments, R³ is—C(═O)NHR^(b), and R^(b) is optionally substituted alkyl. In certainembodiments, R³ is —C(═O)NHR^(b), and R^(b) is optionally substitutedalkenyl. In certain embodiments, R³ is —C(═O)NHR^(b), and R^(b) isoptionally substituted carbocyclyl, heterocyclyl, aryl, or heteroaryl.In certain embodiments, R³ is optionally substituted vinylcarbonyl(e.g., —C(═O)CH═CH₂, —C(═O)CMe=CH₂).

In certain embodiments, R³ is —OR^(b), e.g., —OH. In certainembodiments, R³ is —OR^(b), and R^(b) is optionally substituted alkyl.In certain embodiments, R³ is —OR^(b), and R^(b) is unsubstituted C₁₋₆alkyl. In certain embodiments, R³ is —OR^(b), and R^(b) is optionallysubstituted alkenyl. In certain embodiments, R³ is —OR^(b), and R^(b) isoptionally substituted carbocyclyl, optionally substituted heterocyclyl,optionally substituted aryl optionally substituted heteroaryl. Incertain embodiments, R³ is —OR^(b), and R^(b) is unsubstitutedcarbocyclyl, unsubstituted heterocyclyl, unsubstituted arylunsubstituted heteroaryl. In certain embodiments, R³ is —OR^(b), andR^(b) is optionally substituted acyl, e.g., R³ is —OC(═O)R^(b),—OC(═O)OR^(b), or —OC(═O)N(R^(b))₂. In certain embodiments, R³ is—OR^(b), and R^(b) is an oxygen protecting group (e.g., silyl, TBDPS,TBDMS, TIPS, TES, TMS, MOM, THP, t-Bu, Bn, allyl, acetyl, pivaloyl,benzoyl).

In certain embodiments, R³ is —N(R^(b))₂, e.g., —NH₂, —NHR^(b). Incertain embodiments, R³ is —NH(R^(b)), and R^(b) is optionallysubstituted alkyl. In certain embodiments, R³ is —N(R^(b))₂, and atleast one R^(b) is optionally substituted alkyl. In certain embodiments,R³ is —NH(R^(b)), and R^(b) is unsubstituted alkyl. In certainembodiments, R³ is —N(R^(b))₂, and at least one R^(b) is unsubstitutedalkyl. In certain embodiments, R³ is —NHR^(b), and R^(b) is optionallysubstituted carbocyclyl, optionally substituted heterocyclyl, optionallysubstituted aryl, or optionally substituted heteroaryl. In certainembodiments, R³ is —NHR^(b), and R^(b) is unsubstituted carbocyclyl,unsubstituted heterocyclyl, unsubstituted aryl, or unsubstitutedheteroaryl. In certain embodiments, R³ is —NHR^(b), and R^(b) isoptionally substituted acyl, e.g., R³ is —NHC(═O)R^(b), —NHC(═O)OR^(b),or —NHC(═O)NHR^(b). In certain embodiments, R³ is —N(R^(b))₂, and atleast one R^(b) is a nitrogen protecting group (e.g., Bn, Boc, Cbz,Fmoc, trifluoroacetyl, triphenylmethyl, acetyl, Ts). In certainembodiments, R³ is —N(R^(b))₂, and both R^(b) are joined to form anoptionally substituted heterocyclic or optionally substituted heteroarylring. In certain embodiments, R³ is —N(R^(b))₂, and both R^(b) arejoined to form an unsubstituted heterocyclic or unsubstituted heteroarylring.

Synthesis of the Polyphosphoester Block Copolymer

The block copolymers described herein may be prepared by sequentialpolymerization of monomers corresponding to each block. For example,polymerization of propylene oxide, followed by polymerization ofethylene oxide starting at the terminal groups of the polypropyleneoxide, followed by polymerization of phosphoester monomers starting atthe terminal groups of the polypropylene oxide-polyethylene oxidepolymer. In certain embodiments, a poloxamer comprising polyethylene andpolypropylene blocks is treated with a phosphoester monomer precursor.In some embodiments, the precursor is hydroxydioxaphospholane or adioxaphospholane ester. In certain embodiments, the copolymer of Formula(I):

is prepared by contacting a polymer of Formula (P):

with a compound of Formula (A), or a mixture of compounds of Formula(A):

wherein p, q, r, s, t, G¹, G², and Y are as defined herein.

In certain embodiments, the step of contacting a polymer of Formula (P)with a compound of Formula (A) is performed in the presence of acatalyst. In some embodiments, the catalyst is an organocatalyst. Insome embodiments, the catalyst is a base. In some embodiments, thecatalyst is an organic base. In some embodiments, the catalyst is anon-nucleophlic base. In some embodiments, the catalyst isN,N-diisopropylethylamine (DIPEA, Hünig's base),1,8-diazabicyclo[5.4.0]undec-7-ene (DBU), 2,6-di-tert-butylpyridine, ora phosphazene (e.g., BEMP, t-Bu-P4). In some embodiments, the catalystis 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU).

In some embodiments, the polymer is treated with a compound of Formula(A-i):

A mixture of compounds of Formula (A-i) may also be used, e.g., withdifferent R¹, or a single compound may be used.

In some embodiments, the polymer is treated with a compound Formula(A-ii):

A mixture of compounds of Formula (A-ii) may also be used, e.g., withdifferent R¹, or a single compound may be used.

In some embodiments, the polymer is treated with a mixture of compoundsof Formula (A-i) and (A-ii).

A mixture of compound of Formula (A-i) may also be used, e.g., withdifferent R¹, or a single compound may be used. A mixture of compoundsof Formula (A-ii) may also be used, e.g., with different R¹, or a singlecompound may be used.

In certain embodiments, a first compound of Formula (A) and a secondcompound of Formula (A) are contacted with a copolymer of Formula (P)simultaneously. For example, a compound of Formula (A-i) and a compoundof Formula (A-ii) are added simultaneously to generate polyphosphoesterblocks with a random monomer distribution. In other embodiments, a firstcompound of Formula (A) is contacted with a copolymer of Formula (P),and subsequently a second compound of Formula (A) is contacted with theproduct of contacting a compound of Formula (P) with the first compoundof Formula (A). For example, a compound of Formula (A-i) is added togenerate a first polyphosphoester block with a first monomer (e.g., amonomer of Formula (M-i), and subsequently a compound of Formula (A-ii)is added to generate a second polyphosphoester block with a secondmonomer (e.g., a monomer of Formula (M-ii).

The number of phosphoester monomers (e.g., variables p and t) in theresulting copolymer will be determined by the reaction conditions,reaction time, and the number of equivalents of compound(s) of Formula(A) vs. equivalents of the compound(s) of Formula (P).

In certain embodiments, the copolymer may be further modified afteraddition of the polyphosphoester blocks. In some embodiments, one ormore group Y is modified after polymerization. In some embodiments, oneor more group Y is deprotected after polymerization. In someembodiments, group G¹ or G² is modified after polymerization.

Permeation Enhancers

Permeation enhancer refers to any agent that increases the flux of atherapeutic agent across a barrier (e.g., membrane, layer of cells). Insome embodiments, the barrier is skin. In some embodiments, the barrieris the tympanic membrane. Permeation enhancers may include, but are notlimited to, surfactants (anionic, cationic, nonionic, zwitterionic),terpenes, amino amides, amino esters, azide-containing compounds, andalcohols. Permeation enhancers may include, but are not limited to,surfactants (anionic, cationic, nonionic, zwitterionic), terpenes, aminoamides, amino esters, azide-containing compounds, pyrrolidones,sulfoxides, fatty acids, and alcohols. In certain embodiments, thepermeation enhancer is an anionic surfactant. In certain embodiments,the permeation enhancer is a cation surfactant. In certain embodiments,the permeation enhancer is nonionic surfactant. In certain embodiments,the permeation enhancer is a zwitterionic surfactant. In certainembodiments, the permeation enhancer is a terpene. In certainembodiments, the permeation enhancer is an amino amide. In certainembodiments, the permeation enhancer is an amino ester. In certainembodiments, the permeation enhancer is an azide-containing compound. Incertain embodiments, the permeation enhancer is a pyrrolidone. Incertain embodiments, the permeation enhancer is a sulfoxide. In certainembodiments, the permeation enhancer is a fatty acid. In certainembodiments, the permeation enhancer is an alcohol. In certainembodiments, the permeation enhancer is sodium lauroyl sarcosinate. Incertain embodiments, the permeation enhancer is sorbitan monooleate. Incertain embodiments, the permeation enhancer is octoxynol-9. In certainembodiments, the permeation enhancer is diethyl sebacate. In certainembodiments, the permeation enhancer is sodium polyacrylate (2500000molecular weight (MW)). In certain embodiments, the permeation enhanceris octyldodecanol.

Surfactant permeation enhancers may include, but are not limited to,sodium dodecyl sulfate, ammonium lauryl sulfate, sodium laureth sulfate,cetyl trimethlammonium bromide, cetylpyridinium chloride, benzethoniumchloride, cocamidopropyl betaine, cetyl alcohol, oleyl alcohol, octylglucoside, decyl maltoside, sodium octyl sulfate, sodium decyl sulfate,sodium tetradecyl sulfate, sodium heptadecyl sulfate, sodium eicosylsulfate, nicotine sulfate, sodium taurocholic sulfate, dimethylsulfoxide, sodium tridecyl phosphate; decyldimethyl ammonio propanesulfonate, chembetaine oleyl, myristyldimethyl ammonio propanesulfonate; benzyl pyridinium chloride, dodecyl pyridinium chloride,cetyl pyridinium chloride, benzyldimethyl dodecyl ammonium chloride,benzyldimethyl dodecyl ammonium chloride, benzyldimethyl myristylammonium chloride, benzyldimethyl stearyl ammonium chloride,octyltrimethylammonium bromide, dodecyltrimethylammonium bromide,Polysorbate 20, Polysorbate 40, Polysorbate 60, Polysorbate 80, andbenzalkonium chlorides. In some embodiments, the permeation enhancer issodium dodecyl sulfate, sodium lauryl sulfate, or sodium octyl sulfate.In some embodiments, the permeation enhancer is sodium dodecyl sulfate.In some embodiments, the permeation enhancer is octyl-trimethyl-ammoniumbromide or dodecyl-trimethyl-ammonium bromide. In some embodiments thepermeation enhancer is Polysorbate 20, Polysorbate 40, Polysorbate 60,or Polysorbate 80. In some embodiments the permeation enhancer is abenzalkonium chloride.

In certain embodiments, the permeation enhancer is sodium lauroylsarcosinate, sorbitan monooleate, octoxynol-9, diethyl sebacate, sodiumpolyacrylate (2500000 molecular weight (MW)), or octyldodecanol. Incertain embodiments, the permeation enhancer is sodium lauroylsarcosinate. In certain embodiments, the permeation enhancer is sorbitanmonooleate. In certain embodiments, the permeation enhancer isoctoxynol-9. In certain embodiments, the permeation enhancer is diethylsebacate. In certain embodiments, the permeation enhancer is sodiumpolyacrylate (2500000 molecular weight (MW)). In certain embodiments,the permeation enhancer is octyldodecanol.

In various embodiments, the permeation enhancer is an azone-likecompound. In certain embodiments, the permeation enhancer is a compoundsimilar to azone (e.g., laurocapram) of the formula:

In certain embodiments, the permeation enhancer is1-benzyl-4-(2-((1,1-biphenyl)-4-yloxy)ethyl)piperazine.

In various embodiments, the permeation enhancer is a lipid. In certainembodiments, the lipid used in the composition is selected from thegroup consisting of phosphoglycerides; phosphatidylcholines; dipalmitoylphosphatidylcholine (DPPC); dioleylphosphatidyl ethanolamine (DOPE);dioleyloxypropyltriethylammonium (DOTMA); dioleoylphosphatidylcholine;cholesterol; cholesterol ester; diacylglycerol; diacylglycerolsuccinate;diphosphatidyl glycerol (DPPG); hexanedecanol; fatty alcohols such aspolyethylene glycol (PEG); polyoxyethylene-9-lauryl ether; a surfaceactive fatty acid, such as palmitic acid or oleic acid; fatty acids;fatty acid amides; sorbitan trioleate (Span 85) glycocholate; surfactin;a poloxamer; a sorbitan fatty acid ester such as sorbitan trioleate;lecithin; lysolecithin; phosphatidylserine; phosphatidylinositol;sphingomyelin; phosphatidylethanolamine (cephalin); cardiolipin;phosphatidic acid; cerebrosides; dicetylphosphate;dipalmitoylphosphatidylglycerol; stearylamine; dodecylamine;hexadecyl-amine; acetyl palmitate; glycerol ricinoleate; hexadecylsterate; isopropyl myristate; tyloxapol; poly(ethyleneglycol)5000-phosphatidylethanolamine; and phospholipids. In certainembodiments, the lipid used in the composition is selected from thegroup consisting of phosphoglycerides; phosphatidylcholines; dipalmitoylphosphatidylcholine (DPPC); dioleylphosphatidyl ethanolamine (DOPE);dioleyloxypropyltriethylammonium (DOTMA); dioleoylphosphatidylcholine;cholesterol; cholesterol ester; diacylglycerol; diacylglycerolsuccinate;diphosphatidyl glycerol (DPPG); hexanedecanol; fatty alcohols such aspolyethylene glycol (PEG); polyoxyethylene-9-lauryl ether; a surfaceactive fatty acid, such as palmitic acid or oleic acid; fatty acids;fatty acid amides; sorbitan trioleate (Span 85) glycocholate; surfactin;a poloxamer; a fatty ester (e.g., stearyl methacrylate) a sorbitan fattyacid ester such as sorbitan trioleate; lecithin; lysolecithin;phosphatidylserine; phosphatidylinositol; sphingomyelin;phosphatidylethanolamine (cephalin); cardiolipin; phosphatidic acid;cerebrosides; dicetylphosphate; dipalmitoylphosphatidylglycerol;stearylamine; dodecylamine; hexadecyl-amine; acetyl palmitate; glycerolricinoleate; hexadecyl sterate; isopropyl myristate; tyloxapol;poly(ethylene glycol)5000-phosphatidylethanolamine; and phospholipids.In certain embodiments, the permeation enhancer is a fatty ester. Incertain embodiments, the permeation enhancer is stearyl methacrylate.The lipid may be positively charged, negatively charged, or neutral. Incertain embodiments, the lipid is a combination of lipids. Phospholipidsuseful in the inventive compositions include negatively chargedphosphatidyl inositol, phosphatidyl serine, phosphatidyl glycerol,phosphatic acid, diphosphatidyl glycerol, poly(ethyleneglycol)-phosphatidyl ethanolamine, dimyristoylphosphatidyl glycerol,dioleoylphosphatidyl glycerol, dilauryloylphosphatidyl glycerol,dipalmitotylphosphatidyl glycerol, distearyloylphosphatidyl glycerol,dimyristoyl phosphatic acid, dipalmitoyl phosphatic acid, dimyristoylphosphitadyl serine, dipalmitoyl phosphatidyl serine, phosphatidylserine, and mixtures thereof. Useful zwitterionic phospholipids includephosphatidyl choline, phosphatidyl ethanolamine, sphingomyeline,lecithin, lysolecithin, lysophatidylethanolamine, cerebrosides,dimyristoylphosphatidyl choline, dipalmitotylphosphatidyl choline,distearyloylphosphatidyl choline, dielaidoylphosphatidyl choline,dioleoylphosphatidyl choline, dilauryloylphosphatidyl choline,1-myristoyl-2-palmitoyl phosphatidyl choline, 1-palmitoyl-2-myristoylphosphatidyl choline, 1-palmitoyl-phosphatidyl choline,1-stearoyl-2-palmitoyl phosphatidyl choline, dimyristoyl phosphatidylethanolamine, dipalmitoyl phosphatidyl ethanolamine, brainsphingomyelin, dipalmitoyl sphingomyelin, distearoyl sphingomyelin, andmixtures thereof. Zwitterionic phospholipids constitute any phospholipidwith ionizable groups where the net charge is zero. In certainembodiments, the lipid is phosphatidyl choline.

Exemplary surfactants include, but are not limited to, sodium dioctylsulfo succinate, sodium dodecyl sulfate, cocoamidopropyl betaine, andsodium laureth sulfate, alkyl and alkyl ether sulfates (e.g., sodiumcoconut alkyl triethylene glycol ether sulfate; lithium tallow alkyltriethylene glycol ether sulfate; sodium tallow alkyl hexaoxyethylenesulfate), succinamates, sulfosuccinamates (e.g., disodiumN-octadecyl-sulfosuccinamate, tetrasodiumN-(1,2-dicarboxyethyl)-N-octadecylsulfosuccinamate, diamyl ester ofsodium sulfosuccinic acid, dihexyl ester of sodium sulfosuccinic acid,dioctyl esters of sodium sulfosuccinic acid), olefin sulfonates,hydroxy-alkanesulfonates, beta-alkyloxy alkane sulfonates (e.g.,potassium-β-methoxydecanesulfonate, sodium 2-methoxytridecanesulfonate,potassium 2-ethoxytetradecylsulfonate, sodium2-isopropoxyhexadecylsulfonate, lithium 2-t-butoxytetradecylsulfonate,sodium β-methoxyoctadecysulfonate, ammoniumβ-n-propoxy-dodecylsulfonate), dioctyl esters of sodium sulfosuccinicacid, alkyl ethoxylated sulfates, alkyl sulfates, aliphatic secondaryand tertiary amines (e.g., sodium 3-dodecylaminopropionate,N-alkyltaurines, stearamido propyl dimethyl amine, diethyl amino ethylstearamide, dimethyl stearamine, dimethyl soyamine, soyamine, myristylamine, tridecyl amine, ethyl stearylamine, N-tallowpropane diamine,ethoxylated (5 moles E.O) stearylamine, dihydroxy ethyl stearylamine,and arachidylbehenylamine), alkyl amphoglycinates (e.g.,cocoamphoglycinate, lauroamphocarboxyglycinate,cocoamphocarboxyglycinate); alkyl amphopropionates (e.g.,isostearoamphopropionate, cocoamphocarboxypropionic acid); alkylethoxylated sulfates; alkyl sulfates; aliphatic quaternary ammoniumcompounds (e.g., tallow propane diammonium dichloride,dialkyldimethylammonium chlorides, ditallowdimethyl ammonium chloride,ditallowdimethyl ammonium methyl sulfate, dihexadecyl dimethyl ammoniumchloride, di(hydrogenated tallow) dimethyl ammonium chloride,dioctadecyl dimethyl ammonium chloride, dieicosyl dimethyl ammoniumchloride, didocosyl dimethyl ammonium chloride, di(hydrogenated tallow)dimethyl ammonium acetate, dihexadecyl dimethyl ammonium chloride,dihexadecyl dimethyl ammonium acetate, ditallow dipropyl ammoniumphosphate, ditallow dimethyl ammonium nitrate, and di(coconutalkylbenzyl ammonium chloride); aliphatic phosphonium compounds, aliphaticsulfonium compounds, alkyl amino sulfonates, alkyl betaines (e.g., cocodimethyl carboxymethyl betaine, lauryl dimethyl carboxymethyl betaine,lauryl dimethyl alphacarboxyethyl betaine, cetyl dimethyl carboxymethylbetaine, lauryl bis-(2-hydroxyethyl) carboxy methyl betaine, stearylbis-(2-hydroxypropyl) carboxymethyl betaine, oleyl dimethylgamma-carboxypropyl betaine, lauryl bis-(2-hydroxypropyl)alpha-carboxyethyl betaine), sulfo betaines (e.g., coco dimethylsulfopropyl betaine, stearyl dimethyl sulfopropyl betaine, lauryldimethyl sulfoethyl betaine, lauryl bis(2-hydroxyethyl) sulfopropylbetaine), alkyl amido betaines,4-[N,N-di(2-hydroxyethyl)-N-octadecylammonio]-butane-1-carboxylate;5-[S-3-hydroxypropyl-S-hexadecylsulfonio]-3-hydroxy-pentanel-sulfate;3-[P,P-diethyl-P-3,6,9-trioxatetradexoxylphosphonio]-2-hydroxy-propane-1-phosphate;3-[N,N-dipropyl-N-3-dodecoxy-2-hydroxypropylammonio]-propane-1-phosphate;3-(N,N-dimethyl-N-hexadecylammonio)propane-1-sulfonate;3-(N,N-dimethyl-N-hexadecylammonio)-2-hydroxy-propane-1-sulfonate;4-[N,N-di-(2-hydroxy-ethyl)-N-(2-hydroxydodecyl)ammonio]-butane-1-carboxylate;3-[S-ethyl-S-(3-dodecoxy-2-hydroxypropyl)sulfonio]-propane-1-phosphate;3-[P,P-dimethyl-P-dodecylphosphonio]-propane-1-phosphonate; and5-[N,N-di(3-hydroxypropyl)-N-hexadecylammonio]-2-hydroxypentane-1-sulfate,sodium 3-dodecylaminopropane sulfonate; alkyl amphosulfonates; alkylamphosulfosuccinates; oleoamphopropylsulfonate, andcocoamphopropylsulfonate; polyethylene oxide condensates; long chaintertiary phosphine oxides; long chain dialkyl sulfoxides; Siliconecopolyols (e.g., dimethicone copolyols), stearamide diethanolamide(DEA), cocamide monoethanolamide (MEA), glyceryl monoleate, sucrosestearate, Cetheth-2, Poloxamer 181, hydrogenated tallow amide DEA,polyoxyethylene 4 sorbitol beeswax derivative (ATLAS 6-1702),polyoxyethylene 2 cetyl ether (BRIJ 52), polyoxyethylene 2 stearyl ether(BRIJ 72), polyoxyethylene 2 oleyl ether (BRIJ 92), polyoxyethylene 2oleyl ether (BRIJ 93), sorbitan monopalmitate (SPAN 40), sorbitanmonostearate (SPAN 60), sorbitan tristearate (SPAN 65), sorbitanmonoleate, NF (SPAN 80) sorbitan trioleate (SPAN 85), fluorinated alkylquaternary ammonium iodide; mixed mono- and bis-perfluoroalkylphosphates, ammonium salts; mixed mono- and bis-fluoroalkyl phosphate,ammonium salts, complexed with aliphatic quaternary methosulfates;perfluoroalkyl sulfonic acid, ammonium salts; mixed telomer phosphatediethanolamine salts; amine perfluoroalkyl sulfonates; ammoniumperfluoroalkyl sulfonates; potassium perfluoroalkyl sulfonates;potassium fluorinated alkyl carboxylates; ammonium perfluoroalkylsulfonates; and ammonium perfluoroalkyl carboxylates; sodium dioctylsulfosuccinate; magnesium dioctyl sulfosuccinate; ammonium dioctylsulfosuccinate; magnesium dodecyl sulfate; ammonium dodecyl sulfate;cocoamidopropyl betaine sodium dinonyl sulfo succinate; sodium alphaolefin sulfonate; sodium laureth sulfate; magnesium laureth sulfate;ammonium laureth sulfate; cocoamidopropyl betaine; polyethoxylatedglycol ether of glyceryl isostewarate; polyethoxylated glycol ether ofglyceryl monooleate; PEG-30 glyceryl isostearate; polyoxyethyleneglycerol monoleate; polyethylene glycol; PPG-18; PPG-10; 18 dimethicone;1 dimethicon; cetyl polyethylene glycol; glyceryl monostearate;laureth-23; and PEG 75 lanolin. In certain embodiments, the surfactantis a silicon-containing chemical compound. Exemplary silicon-baseddetergents, emulsifiers, or surfactants useful in cosmetic compositionsinclude dimethicone, cyclopentasiloxane, cyclohexasiloxane,PEG/dimethicone copolymers, PPG/dimethicone copolymers,phenyltrimethicone, alkyl silicones, amodimethicone, siliconequaternium-18, and dimethiconol.

Terpene permeation enhancers may include, but are not limited to,limonene, cymene, pinene, camphor, menthol, comphone, phellandrine,sabinene, terpinene, borneol, cineole, geraniol, linalol, pipertone,terpineol, eugenol, eugenol acetate, safrole, benzyl benzoate, humulene,beta-caryophylene, eucakytol, hexanoic acid, octanoic acid, decanoicacid, undecanoic acid, dodecanoic acid, tridecanoic acid, myristic acid,palmitic acid, stearic acid, oleic acid, linoleic acid, linolenic acid,cholic acid; ethyl undecanoate, methyl laurate, methyl myristate,isopropyl myristate, isopropyl palmitate, palmityl palmitate, diethylsebaccate, glyceryl monolaurate, glyceryl monooleate, andethylpiperazine carboxylate. Any terpene or terpeniod compound may beused as a permeation enhancer in the inventive compositions. In certainembodiments, the permeation enhancer is limonene.

Alcohol permeation enhancers may include, but are not limited to,methanol, ethanol, propanol, isopropanol, butanol, isobutyl alcohol, andtert-amyl alcohol. In certain embodiments, the permeation enhancer is acompound with more than one hydroxyl group (e.g., glycerol). Forexample, the permeation enhancer may contain two, three, four, five, ormore hydroxyl groups. In certain embodiments, the permeation enhancer isa hydroxyl-containing polymer.

In certain embodiments, an amino amide or amino ester permeationenhancers is an anesthetic agent. Amino amide and amino ester permeationenhancers may include, but are not limited to bupivicaine, tetracaine,procaine, proparacaine, propoxycaine, dimethocaine, cyclomethycaine,chloroprocaine, benzocaine, lidocaine, prilocaine, levobupivicaine,ropivacaine, dibucaine, articaine, carticaine, etidocaine, mepivacaine,piperocaine, and trimecaine. In certain embodiments, the permeationenhancer is bupivacaine.

In certain embodiments, the composition comprises a combination ofpermeation enhancers. In certain embodiments, the combination comprisespermeation enhancers of the same type (e.g., both surfactants, bothterpenes). In certain embodiments, the combination comprises permeationenhancers of different types (e.g., a surfactant and a terpene). Incertain embodiments, combination comprises a surfactant and a terpene.In certain embodiments, the combination comprises a cationic surfactantand a terpene. In certain embodiments, the combination comprises ananionic surfactant and a terpene. In certain embodiments, thecombination comprises a nonionic or zwitterionic surfactant and aterpene.

In certain embodiments, the combination comprises a surfactant and anamino amide or amino ester. In certain embodiments, the combinationcomprises a cationic surfactant and an amino amide or amino ester. Incertain embodiments, the combination comprises an anionic surfactant andan amino amide or amino ester. In certain embodiments, the combinationcomprises a nonionic or zwitterionic surfactant and an amino amide oramino ester. In certain embodiments, the combination comprises is aterpene and an amino amide or amino ester. In some embodiments, theamino amide or amino ester is an anesthetic agent. In some embodiments,the anesthetic agent is bupivacaine.

In some embodiments, the permeation enhancer is a combination ofcompounds selected from two or three of groups (i) to (iii):

-   -   (i) a surfactant selected from: sodium dodecyl sulfate, ammonium        lauryl sulfate, sodium laureth sulfate, cetyl trimethlammonium        bromide, cetylpyridinium chloride, benzethonium chloride,        cocamidopropyl betaine, cetyl alcohol, oleyl alcohol, octyl        glucoside, decyl maltoside, sodium octyl sulfate, sodium decyl        sulfate, sodium tetradecyl sulfate, sodium heptadecyl sulfate,        sodium eicosyl sulfate, nicotine sulfate, sodium taurocholic        sulfate, dimethyl sulfoxide, sodium tridecyl phosphate;        decyldimethyl ammonio propane sulfonate, chembetaine oleyl,        myristyldimethyl ammonio propane sulfonate; benzyl pyridinium        chloride, dodecyl pyridinium chloride, cetyl pyridinium        chloride, benzyldimethyl dodecyl ammonium chloride,        benzyldimethyl dodecyl ammonium chloride, benzyldimethyl        myristyl ammonium chloride, benzyldimethyl stearyl ammonium        chloride, octyltrimethylammonium bromide,        dodecyltrimethylammonium bromide, Polysorbate 20, Polysorbate        40, Polysorbate 60, Polysorbate 80, and benzalkonium chlorides;    -   (ii) a terpene selected from: limonene, cymene, pinene, camphor,        menthol, comphone, phellandrine, sabinene, terpinene, borneol,        cineole, geraniol, linalol, pipertone, terpineol, eugenol,        eugenol acetate, safrole, benzyl benzoate, humulene,        beta-caryophylene, eucakytol, hexanoic acid, octanoic acid,        decanoic acid, undecanoic acid, dodecanoic acid, tridecanoic        acid, myristic acid, palmitic acid, stearic acid, oleic acid,        linoleic acid, linolenic acid, cholic acid; ethyl undecanoate,        methyl laurate, methyl myristate, isopropyl myristate, isopropyl        palmitate, palmityl palmitate, diethyl sebaccate, glyceryl        monolaurate, glyceryl monooleate, or ethylpiperazine        carboxylate;    -   (iii) and an anesthetic selected from: bupivicaine, tetracaine,        procaine, proparacaine, propoxycaine, dimethocaine,        cyclomethycaine, chloroprocaine, benzocaine, lidocaine,        prilocaine, levobupivicaine, ropivacaine, dibucaine, articaine,        carticaine, etidocaine, mepivacaine, piperocaine, and        trimecaine.        In some embodiments, permeation enhancer is a combination of        compounds from at least two of the groups (i) to (iii), listed        above, and includes sodium octyl sulfate, sodium dodecyl        sulfate, octyl trimethylammonium bromide, dodecyl        trimethylammonium bromide, Polysorbate 20, or Polysorbate 80 as        a surfactant. In some embodiments, permeation enhancer is a        combination of compounds from at least two of the groups (i) to        (iii), listed above, and includes sodium dodecyl sulfate as a        surfactant. In some embodiments, permeation enhancer is a        combination of compounds from at least two of the groups (i)        to (iii) listed above, and includes limonene as a surfactant. In        some embodiments, permeation enhancer is a combination of        compounds from at least two of the groups (i) to (iii), listed        above, and includes bupivacaine as an anesthetic. In some        embodiments, permeation enhancer is a combination of compounds        from at least two of the groups (i) to (iii), listed above, and        includes sodium dodecyl sulfate, octyl trimethylammonium        bromide, dodecyl trimethylammonium bromide, Polysorbate 20, or        Polysorbate 80 as a surfactant, and limonene as a terpene. In        some embodiments, permeation enhancer is a combination of        compounds from at least two of the groups (i) to (iii), listed        above, and includes sodium dodecyl sulfate as a surfactant, and        limonene as a terpene. In some embodiments, permeation enhancer        is a combination of compounds from at least two of the        groups (i) to (iii), listed above, and includes sodium dodecyl        sulfate, octyl trimethylammonium bromide, dodecyl        trimethylammonium bromide, Polysorbate 20, or Polysorbate 80 as        a surfactant, and bupivacaine as an anesthetic. In some        embodiments, permeation enhancer is a combination of compounds        from at least two of the groups (i) to (iii), listed above, and        includes sodium dodecyl sulfate as a surfactant, and bupivacaine        as an anesthetic. In some embodiments, permeation enhancer is a        combination of compounds from at least two of the groups (i) to        (iii), listed above, and includes limonene as a terpene, and        bupivacaine as an anesthetic. In some embodiments, permeation        enhancer is a combination of compounds from at least two of the        groups (i) to (iii), listed above, and includes sodium dodecyl        sulfate, octyl trimethylammonium bromide, dodecyl        trimethylammonium bromide, Polysorbate 20, or Polysorbate 80 as        a surfactant, limonene as a terpene, and bupivacaine as an        anesthetic. In some embodiments, permeation enhancer is a        combination of compounds from at least two of the groups (i) to        (iii), listed above, and includes sodium dodecyl sulfate as a        surfactant, limonene as a terpene, and bupivacaine as an        anesthetic.

In certain embodiments, the percent weight of permeation enhancer in thecomposition is between about 0.1% to about 1%, between about 1% to about3%, or between about 3% to about 10%. In certain embodiments, thepercent weight of permeation enhancer in the composition is betweenabout 0.1% to about 1%. In certain embodiments, the percent weight ofpermeation enhancer in the composition is between about 1% to about 3%.In certain embodiments, the percent weight of permeation enhancer in thecomposition is between about 0.1% to about 10%. In certain embodiments,the percent weight of permeation enhancer in the composition is between0.1% to about 1%, between about 1% to about 2%, between about 2% toabout 3%, between about 3% to about 4%, between about 4% to about 5%,between about 5% to about 6%, between about 6% to about 7%, betweenabout 7% to about 8%, between about 8% to about 9%, or between about 9%to about 10%.

In some embodiments, the percent weight of sodium dodecyl sulfate in thecomposition is between about 0.1% to about 3%. In some embodiments, thepercent weight in the composition of sodium dodecyl sulfate is about 1%.In some embodiments, the percent weight of bupivicaine in thecomposition is between about 0.1 to about 3%. In some embodiments, thepercent weight in the composition of bupivicaine is about 0.5%. In someembodiments, the percent weight of limonene in the composition isbetween about 0.1% to about 3%. In some embodiments, the percent weightin the composition of limonene is about 0.5%.

Therapeutic Agents

A therapeutic agent can be any agent used to treat any ear disease, orsymptom of an ear disease. Therapeutic agents may include antimicrobialagents. Therapeutic agents may include, but are not limited to,antimicrobial agents, antibiotics, anesthetics, anti-inflammatories,analgesics, anti-fibrotics, anti-sclerotics, and anticoagulants.Therapeutic agents may include, but are not limited to, antibiotics,anesthetics, anti-inflammatories, analgesics, anti-fibrotics,anti-sclerotics, and anticoagulants. In certain embodiments, thetherapeutic agent is an antimicrobial agent. In certain embodiments, thetherapeutic agent is an antibiotic agent. In certain embodiments, thetherapeutic agent is an anesthetic agent. In certain embodiments, thetherapeutic agent is an anti-inflammatory agent. In certain embodiments,the therapeutic agent is an analgesic agent. In certain embodiments, thetherapeutic agent is an anti-fibrotic agent. In certain embodiments, thetherapeutic agent is an anti-sclerotic agent. In certain embodiments,the therapeutic agent is an anticoagulant agent.

In various aspects, the therapeutic agents may comprise between about0.01 percent to about 10 percent of the composition. In variousembodiments, the therapeutic agents may comprise between about 0.01percent to about 1 percent of the composition, comprise between about 1percent to about 2 percent of the composition, comprise between about 2percent to about 3 percent of the composition, comprise between about 3percent to about 4 percent of the composition, comprise between about 4percent to about 5 percent of the composition, comprise between about 5percent to about 6 percent of the composition, comprise between about 6percent to about 7 percent of the composition, comprise between about 7percent to about 8 percent of the composition, comprise between about 8percent to about 9 percent of the composition, or comprise between about9 percent to about 10 percent of the composition.

The exact amount required will vary from subject to subject, dependingon the species, age, and general condition of the subject, theparticular compound, its mode of administration, its mode of activity,condition being treated, and the like. The compositions described hereinare preferably formulated in dosage unit form for ease of administrationand uniformity of dosage. It will be understood, however, that the totaldaily usage of the compounds and compositions will be decided by theattending physician within the scope of sound medical judgment. Thespecific therapeutically effective dose level for any particular patientor organism will depend upon a variety of factors including the disorderbeing treated and the severity of the disorder; the activity of thespecific compound employed; the specific composition employed; the age,body weight, general health, sex and diet of the patient; the time ofadministration, route of administration, and rate of excretion of thespecific compound employed; the duration of the treatment; drugs used incombination or coincidental with the specific compound employed; andlike factors well known in the medical arts.

In certain embodiments, the therapeutic agent is an antimicrobial agent.In certain embodiments, the therapeutic agent is an antibiotic. Anyantibiotic may be used in the inventive system. In certain embodimentsthe antibiotic is approved for use in humans or other animals. Incertain embodiments the antibiotic is approved for use by the U.S. Food& Drug Administration. In certain embodiments, the antibiotic may beselected from the group consisting of cephalosporins, quinolones,polypeptides, macrolides, penicillins, and sulfonamides. Exemplaryantibiotics may include, but are not limited to, ciprofloxacin,cefuroxime, cefadroxil, cefazolin, cefalotin, cefalexin, cefaclor,cefamandole, cefoxitin, cefprozil, cefuroxime, cefixime, cefdinir,cefditoren, cefoperazone, cefotaxime, cefpodoxime, ceftazidime,ceftibuten, ceftizoxime, ceftriaxone, cefepime, ceftobiprole, enoxacin,gatifloxacin, levofloxacin, lomefloxacin, moxifloxacin, norfloxacin,ofloxacin, trovafloxacin, bacitracin, colistin, polymyxin B,azithromycin, clarithromycin, dirithromycin, erythromycin,roxithromycin, troleandomycin, telithromycin, spectinomycin,amoxicillin, ampicillin, azlocillin, carbenicillin, cloxacillin,dicloxacillin, flucloxacillin, mezlocillin, meticillin, nafcillin,oxacillin, penicillin, piperacillin, ticarcillin, mafenide,sulfacetamide, sulfamethizole, sulfasalazine, sulfisoxazole,trimethoprim, and trimethoprim-sulfamethoxazole.

In certain embodiments, the antibiotic is a quinolone. In certainembodiments, the antibiotic is a carbapenem. In certain embodiments, theantibiotic is In certain embodiments, the antibiotic is amoxicillin,azithromicicn, cefuroxime, ceftriaxone, trimethoprim, levofloxacin,moxifloxacin, meropenem, or ciprofloxacin. In some embodiments, theantibiotic is ciprofloxacin. In some embodiments, the antibiotic isciprofloxacin and pharmaceutically acceptable salts thereof. In someembodiments, the antibiotic is ciprofloxacin hydrochloride. In someembodiments, the antibiotic is levofloxacin.

Exemplary antibiotics, include, but are not limited to: Abamectin,Actinomycin (e.g., Actinomycin A, Actinomycin C, Actinomycin D,Aurantin), Alatrofloxacin mesylate, Amikacin sulfate, Aminosalicylicacid, Anthracyclines (e.g., Aclarubicin, Adriamycin, Doxorubicin,Epirubicin, Idarubicin), Antimycin (e.g., Antimycin A), Avermectin, BAL30072, Bacitracin, Bleomycin, Cephalosporins (e.g.,7-Aminocephalosporanic acid, 7-Aminodeacetoxycephalo sporanic acid,Cefaclor, Cefadroxil, Cefamandole, Cefazolin, Cefepime, Cefixime,Cefmenoxime, Cefmetazole, Cefoperazone, Cefotaxime, Cefotetan, Cefotiam,Cefoxitin, Cefpirome, Cefpodoxime proxetil, Cefsulodin, Cefsulodinsodium, Ceftazidime, Ceftizoxime, Ceftriaxone, Cefuroxime, Cephalexin,Cephaloridine, Cephalosporin C, Cephalothin, Cephalothin sodium,Cephapirin, Cephradine), Ciprofloxacin, Enrofloxacin, Clarithromycin,Clavulanic acid, Clindamycin, Colicin, Cyclosporin (e.g. Cyclosporin A),Dalfopristin/quinupristin, Daunorubicin, Doxorubicin, Epirubicin, GSK1322322, Geneticin, Gentamicin, Gentamicin sulfate, Gramicidin (e.g.Gramicidin A), Grepafloxacin hydrochloride, Ivermectin, Kanamycin (e.g.Kanamycin A), Lasalocid, Leucomycin, Levofloxacin, Linezolid,Lomefloxacin, Lovastatin, MK 7655, Meropenem, Mevastatin, Mithramycin,Mitomycin, Monomycin, Natamycin, Neocarzinostatin, Neomycin (e.g.Neomycin sulfate), Nystatin, Oligomycin, Olivomycin, Pefloxacin,Penicillin (e.g. 6-Aminopenicillanic acid, Amoxicillin,Amoxicillin-clavulanic acid, Ampicillin, Ampicillin sodium, Azlocillin,Carbenicillin, Cefoxitin, Cephaloridine, Cloxacillin, Dicloxacillin,Mecillinam, Methicillin, Mezlocillin, Nafcillin, Oxacillin, PenicillinG, Penicillin G potassium, Penicillin G procaine, Penicillin G sodium,Penicillin V, Piperacillin, Piperacillin-tazobactam, Sulbactam,Tazobactam, Ticarcillin), Phleomycin, Polymyxin (e.g., Colistin,Polymyxin B), Pyocin (e.g. Pyocin R), RPX 7009, Rapamycin, Ristocetin,Salinomycin, Sparfloxacin, Spectinomycin, Spiramycin, Streptogramin,Streptovaricin, Tedizolid phosphate, Teicoplanin, Telithromycin,Tetracyclines (e.g. Achromycin V, Demeclocycline, Doxycycline,Doxycycline monohydrate, Minocycline, Oxytetracycline, Oxytetracyclinehydrochloride Tetracycline, Tetracycline hydrochloride), Trichostatin A,Trovafloxacin, Tunicamycin, Tyrocidine, Valinomycin, (−)-Florfenicol,Acetylsulfisoxazole, Actinonin, Amikacin sulfate, Benzethonium chloride,Cetrimide, Chelerythrine, Chlorhexidine (e.g., Chlorhexidine gluconate),Chlorhexidine acetate, Chlorhexidine gluconate, Chlorothalonil,Co-Trimoxazole, Dichlorophene, Didecyldimethylammonium chloride,Dihydrostreptomycin, Enoxacin, Ethambutol, Fleroxacin, Furazolidone,Methylisothiazolinone, Monolaurin, Oxolinic acid, Povidone-iodine,Spirocheticides (e.g., Arsphenamine, Neoarsphenamine), Sulfaquinoxaline,Thiamphenicol, Tinidazole, Triclosan, Trovafloxacin, Tuberculostatics(e.g., 4-Aminosalicylic acid, AZD 5847, Aminosalicylic acid,Ethionamide), Vidarabine, Zinc pyrithione, and Zirconium phosphate.

In certain embodiments, the therapeutic agent is an Food and DrugAdministration (FDA) approved drug for treating infections or infectiousdiseases. Exemplary FDA approved agents include, but are not limited to:Avycaz (ceftazidime-avibactam), Cresemba (isavuconazonium sulfate),Evotaz (atazanavir and cobicistat, Prezcobix (darunavir and cobicistat),Dalvance (dalbavancin), Harvoni (ledipasvir and sofosbuvir), Impavido(miltefosine), Jublia (efinaconazole), Kerydin (tavaborole),Metronidazole, Orbactiv (oritavancin), Rapivab (peramivir injection),Sivextro (tedizolid phosphate), Triumeq (abacavir, dolutegravir, andlamivudine), Viekira Pak (ombitasvir, paritaprevir, ritonavir anddasabuvir), Xtoro (finafloxacin), Zerbaxa (ceftolozane+tazobactam), Luzu(luliconazole), Olysio (simeprevir), Sitavig (acyclovir), Sovaldi(sofosbuvir), Abthrax (raxibacumab), Afinitor (everolimus), Cystaran(cysteamine hydrochloride), Dymista (azelastine hydrochloride andfluticasone propionate), Fulyzaq (crofelemer), Jetrea (ocriplasmin),Linzess (linaclotide), Qnasl (beclomethasone dipropionate) nasalaerosol, Sirturo (bedaquiline), Sklice (ivermectin), Stribild(elvitegravir, cobicistat, emtricitabine, tenofovir disoproxilfumarate), Tudorza Pressair (aclidinium bromide inhalation powder),Complera (emtricitabine/rilpivirine/tenofovir disoproxil fumarate),Dificid (fidaxomicin), Edurant (rilpivirine), Eylea (aflibercept),Firazyr (icatibant), Gralise (gabapentin), Incivek (telaprevir),Victrelis (boceprevir), Egrifta (tesamorelin), Teflaro (ceftarolinefosamil), Zymaxid (gatifloxacin), Bepreve (bepotastine besilate),Vibativ (telavancin), Aptivus (tipranavir), Astepro (azelastinehydrochloride nasal spray), Intelence (etravirine), Patanase(olopatadine hydrochloride), Viread (tenofovir disoproxil fumarate),Isentress (raltegravir), Selzentry (maraviroc), Veramyst (fluticasonefuroate), Xyzal (levocetirizine dihydrochloride), Eraxis(anidulafungin), Noxafil (posaconazole), Prezista (darunavir), Tyzeka(telbivudine), Veregen (kunecatechins), Baraclude (entecavir), Fuzeon(enfuvirtide), Lexiva (fosamprenavir calcium), Reyataz (atazanavirsulfate), Clarinex, Hepsera (adefovir dipivoxil), Pegasys (peginterferonalfa-2a), Sustiva, Vfend (voriconazole), Zelnorm (tegaserod maleate),Avelox (moxifloxacin hydrochloride), Cancidas, Invanz, Peg-Intron(peginterferon alfa-2b), Rebetol (ribavirin), Spectracef, Tavist(clemastine fumarate), Twinrix, Valcyte (valganciclovir HCl), Xigris(drotrecogin alfa), ABREVA (docosanol), Cefazolin, Kaletra, Lamisil(terbinafine hydrochloride), Lotrisone (clotrimazole/betamethasonediproprionate), Lotronex (alosetron HCL), Trizivir (abacavir sulfate,lamivudine, zidovudine AZT), Synercid, Synagis, Viroptic, Aldara(imiquimod), Bactroban, Ceftin (cefuroxime axetil), Combivir, Condylox(pokofilox), Famvir (famciclovir), Floxin, Fortovase, INFERGEN(interferon alfacon-1), Intron A (interferon alfa-2b, recombinant),Mentax (butenafine HCl), Norvir (ritonavir), Omnicef, Rescriptor(delavirdine mesylate), Taxol, Timentin, Trovan, VIRACEPT (nelfinavirmesylate), Zerit (stavudine), AK-Con-A (naphazoline ophthalmic), Allegra(fexofenadine hydrochloride), Astelin nasal spray, Atrovent (ipratropiumbromide), Augmentin (amoxicillin/clavulanate), Crixivan (Indinavirsulfate), Elmiron (pentosan polysulfate sodium), Havrix, Leukine(sargramostim), Merrem (meropenem), Nasacort AQ (triamcinoloneacetonide), Tavist (clemastine fumarate), Vancenase AQ, Videx(didanosine), Viramune (nevirapine), Zithromax (azithromycin), Cedax(ceftibuten), Clarithromycin (Biaxin), Epivir (lamivudine), Invirase(saquinavir), Valtrex (valacyclovir HCl), Zyrtec (cetirizine HCl),Acyclovir, Penicillin (penicillin g potassium), Cubicin (Daptomycin),Factive (Gemifloxacin), Albenza (albendazole), Alinia (nitazoxanide),Altabax (retapamulin), AzaSite (azithromycin), Besivance (besifloxacinophthalmic suspension), Biaxin XL (clarithromycin extended-release),Cayston (aztreonam), Cleocin (clindamycin phosphate), Doribax(doripenem), Dynabac, Flagyl ER, Ketek (telithromycin), Moxatag(amoxicillin), Rapamune (sirolimus), Restasis (cyclosporine), Tindamax(tinidazole), Tygacil (tigecycline), and Xifaxan (rifaximin).

In certain embodiments, the therapeutic agent is an anesthetic. Anyanesthetic may be used in the inventive system. In certain embodimentsthe anesthetic is approved for use in humans or other animals. Incertain embodiments the anesthetic is approved for use by the U.S. Food& Drug Administration. Exemplary anesthetics may include, but are notlimited to bupivicaine, tetracaine, procaine, proparacaine,propoxycaine, dimethocaine, cyclomethycaine, chloroprocaine, benzocaine,lidocaine, prilocain, levobupivicaine, ropivacaine, dibucaine,articaine, carticaine, etidocaine, mepivacaine, piperocaine, andtrimecaine. In certain embodiments, the anesthetic is bupivicaine.

In certain embodiments, the therapeutic agent is an anti-inflammatoryagent. The anti-inflammatory agent may be a non-steroidalanti-inflammatory agent or a steroidal anti-inflammatory agent. Incertain embodiments, the therapeutic agent is a steroidalanti-inflammatory agent. In certain embodiments, the therapeutic agentis a steroid. Exemplary anti-inflammatory agents may include, but arenot limited to, acetylsalicylic acid, amoxiprin, benorylate/benorilate,choline magnesium salicylate, diflunisal, ethenzamide, faislamine,methyl salicylate, magnesium salicylate, salicyl salicylate,salicylamide, diclofenac, aceclofenac, acemetacin, alclofenac,bromfenac, etodolac, indometacin, nabumetone, oxametacin, proglumetacin,sulindac, tolmetin, ibuprofen, alminoprofen, benoxaprofen, carprofen,dexibuprofen, dexketoprofen, fenbufen, fenoprofen, flunoxaprofen,flurbiprofen, ibuproxam, indoprofen, ketoprofen, ketorolac, loxoprofen,naproxen, oxaprozin, pirprofen, suprofen, tiaprofenic acid, mefenamicacid, flufenamic acid, meclofenamic acid, tolfenamic acid,phenylbutazone, ampyrone, azapropazone, clofezone, kebuzone, metamizole,mofebutazone, oxyphenbutazone, phenazone, phenylbutazone,sulfinpyrazone, piroxicam, droxicam, lornoxicam, meloxicam, tenoxicam,hydrocortisone, cortisone acetate, prednisone, prednisolone,methylprednisolone, dexamethasone, betamethasone, triamcinolone,beclometasone, fludrocortisone acetate, deoxycorticosterone acetate, andaldosterone.

In various embodiments, combinations of various permeation enhancers andtherapeutic agents have been observed to have a synergistic andheightened efficacy. In various aspects, such combinations may include,but are not limited to, ciprofloxacin and limonene. In various aspects,such combinations may include, but are not limited to, ciprofloxacin andsodium dodecyl sulfate. In various aspects such combinations mayinclude, but are not limited to, sodium dodecyl sulfate, limonene,bupivacaine, and ciprofloxacin. In various aspects, such combination mayinclude, but are not limited to, sodium dodecyl sulfate, limonene andciprofloxacin.

In another aspect, provided herein are pharmaceutical compositionscomprising at least one of the compounds as described herein, or apharmaceutically acceptable derivative thereof. In certain embodiments,the pharmaceutical composition includes a combination of therapeuticagents. In certain embodiments, the composition includes an antibioticand an additional therapeutic agent. In certain embodiments, thecomposition includes an antibiotic agent and an anti-inflammatory agent.In other embodiments, the composition includes an antibiotic agent andan anesthetic agent. In certain embodiments, the composition includesmore than one antibiotic agent.

In certain embodiments, the additional therapeutic agent is ananti-inflammatory agent (e.g., a steroid). In certain embodiments, thefirst therapeutic agent is an antibiotic and the additional therapeuticagent is an anti-inflammatory agent. In certain embodiments, the firsttherapeutic agent is an antibiotic and the additional therapeutic agentis a steroid. Steroids include, but are not limited to, cortisol,hydrocortisone acetate, cortisone acetate, tixocortol pivalate,prednisolone, methylprednisolone, prednisone, triamcinolone acetonide,triamcinolone alcohol, mometasone, amcinonide, budesonide, desonide,fluocinonide, fluocinolone acetonide, halcinonide, betamethasone,betamethasone sodium phosphate, dexamethasone, dexamethasone sodiumphosphate, fluocortolone, hydrocortisone-17-valerate, halometasone,alclometasone dipropionate, betamethasone valerate, betamethasonedipropionate, prednicarbate, clobetasone-17-butyrate,clobetasol-17-propionate, fluocortolone caproate, fluocortolonepivalate, fluprednidene acetate, hydrocortisone-17-butyrate,hydrocortisone-17-aceponate, hydrocortisone-17-buteprate, ciclesonide,and prednicarbate. In some embodiments, the additional anti-inflammatoryagent is dexamethasone.

In certain embodiments, the additional therapeutic agent is aβ-lactamase inhibitor. In certain embodiments, the first therapeuticagent is an antibiotic (e.g., a β-lactam) and the additional therapeuticagent is a β-lactamase inhibitor. β-Lactamase inhibitors include, butare not limited to, avibactam, clavulanic acid, tazobactam, andsulbactam. The β-lactamase inhibitor may be particularly useful incompositions comprising a β-lactam antibiotic. The β-lactamase inhibitormay increase the efficacy of a β-lactam antibiotic or allow for theβ-lactam antibiotic to be present in the composition in a lowerconcentration than for compositions not containing a β-lactamaseinhibitor.

Furthermore, after formulation with an appropriate pharmaceuticallyacceptable carrier in a desired dosage, the pharmaceutical compositionscan be administered to humans and other animals.

Dosage forms include, but are not limited to, pharmaceuticallyacceptable emulsions, microemulsions, solutions, suspensions, syrups,and elixirs. In addition to the active compounds, the liquid dosageforms may contain inert diluents commonly used in the art such as, forexample, water or other solvents, solubilizing agents and emulsifierssuch as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethylacetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butyleneglycol, dimethylformamide, oils (in particular, cottonseed, groundnut,corn, germ, olive, castor, and sesame oils), glycerol,tetrahydrofurfuryl alcohol, polyethylene glycols and fatty acid estersof sorbitan, and mixtures thereof. Besides inert diluents, thecompositions can also include adjuvants such as wetting agents,emulsifying and suspending agents, and perfuming agents. In certainembodiments, the composition comprises a solubilizing agents such anCremophor, alcohols, oils, modified oils, glycols, polysorbates,cyclodextrins, polymers, and combinations thereof.

It will also be appreciated that the compositions described herein canbe employed in combination therapies, that is, the compounds andpharmaceutical compositions can be administered concurrently with, priorto, or subsequent to, one or more other desired therapeutics or medicalprocedures. The particular combination of therapies (therapeutics orprocedures) to employ in a combination regimen will take into accountcompatibility of the desired therapeutics and/or procedures and thedesired therapeutic effect to be achieved. It will also be appreciatedthat the therapies employed may achieve a desired effect for the samedisorder (for example, an inventive compound may be administeredconcurrently with another anticancer agent), or they may achievedifferent effects (e.g., control of any adverse effects).

In certain embodiments, the composition comprises a diagnostic agent. Insome embodiments, the diagnostic agent is a X-ray contrast agent. Insome embodiments, the diagnostic agent comprises a radioactive isotope.In some embodiments, the diagnostic agent is a dye.

Other Additives

In certain embodiments, the composition comprises one or more additionaladditives. For example, an additional additive may be a diluent, bindingagent, preservative, buffering agent, lubricating agent, perfumingagent, antiseptic agent, or oil.

Exemplary diluents include calcium carbonate, sodium carbonate, calciumphosphate, dicalcium phosphate, calcium sulfate, calcium hydrogenphosphate, sodium phosphate lactose, sucrose, cellulose,microcrystalline cellulose, kaolin, mannitol, sorbitol, inositol, sodiumchloride, dry starch, cornstarch, powdered sugar, and mixtures thereof.

Exemplary binding agents include starch (e.g., cornstarch and starchpaste), gelatin, sugars (e.g., sucrose, glucose, dextrose, dextrin,molasses, lactose, lactitol, mannitol, etc.), natural and synthetic gums(e.g., acacia, sodium alginate, extract of Irish moss, panwar gum,ghatti gum, mucilage of isapol husks, carboxymethylcellulose,methylcellulose, ethylcellulose, hydroxyethylcellulose, hydroxypropylcellulose, hydroxypropyl methylcellulose, microcrystalline cellulose,cellulose acetate, poly(vinyl-pyrrolidone), magnesium aluminum silicate(Veegum®), and larch arabogalactan), alginates, polyethylene oxide,polyethylene glycol, inorganic calcium salts, silicic acid,polymethacrylates, waxes, water, alcohol, and/or mixtures thereof.

Exemplary preservatives include antioxidants, chelating agents,antimicrobial preservatives, antifungal preservatives, antiprotozoanpreservatives, alcohol preservatives, acidic preservatives, and otherpreservatives. In certain embodiments, the preservative is anantioxidant. In other embodiments, the preservative is a chelatingagent. In certain embodiments, the preservative is benzalkoniumchloride.

Exemplary antioxidants include alpha tocopherol, ascorbic acid, acorbylpalmitate, butylated hydroxyanisole, butylated hydroxytoluene,monothioglycerol, potassium metabisulfite, propionic acid, propylgallate, sodium ascorbate, sodium bisulfite, sodium metabisulfite, andsodium sulfite.

Exemplary antifungal preservatives include butyl paraben, methylparaben, ethyl paraben, propyl paraben, benzoic acid, hydroxybenzoicacid, potassium benzoate, potassium sorbate, sodium benzoate, sodiumpropionate, and sorbic acid.

Exemplary alcohol preservatives include ethanol, polyethylene glycol,phenol, phenolic compounds, bisphenol, chlorobutanol, hydroxybenzoate,and phenylethyl alcohol.

Exemplary acidic preservatives include vitamin A, vitamin C, vitamin E,beta-carotene, citric acid, acetic acid, dehydroacetic acid, ascorbicacid, sorbic acid, and phytic acid.

Other preservatives include tocopherol, tocopherol acetate, deteroximemesylate, cetrimide, butylated hydroxyanisol (BHA), butylatedhydroxytoluened (BHT), ethylenediamine, sodium lauryl sulfate (SLS),sodium lauryl ether sulfate (SLES), sodium bisulfite, sodiummetabisulfite, potassium sulfite, potassium metabisulfite, Glydant®Plus, Phenonip®, methylparaben, Germall® 115, Germaben® II, Neolone®,Kathon®, and Euxyl®.

Exemplary buffering agents include citrate buffer solutions, acetatebuffer solutions, phosphate buffer solutions, ammonium chloride, calciumcarbonate, calcium chloride, calcium citrate, calcium glubionate,calcium gluceptate, calcium gluconate, D-gluconic acid, calciumglycerophosphate, calcium lactate, propanoic acid, calcium levulinate,pentanoic acid, dibasic calcium phosphate, phosphoric acid, tribasiccalcium phosphate, calcium hydroxide phosphate, potassium acetate,potassium chloride, potassium gluconate, potassium mixtures, dibasicpotassium phosphate, monobasic potassium phosphate, potassium phosphatemixtures, sodium acetate, sodium bicarbonate, sodium chloride, sodiumcitrate, sodium lactate, dibasic sodium phosphate, monobasic sodiumphosphate, sodium phosphate mixtures, tromethamine, magnesium hydroxide,aluminum hydroxide, alginic acid, pyrogen-free water, isotonic saline,Ringer's solution, ethyl alcohol, and mixtures thereof.

Exemplary lubricating agents include magnesium stearate, calciumstearate, stearic acid, silica, talc, malt, glyceryl behanate,hydrogenated vegetable oils, polyethylene glycol, sodium benzoate,sodium acetate, sodium chloride, leucine, magnesium lauryl sulfate,sodium lauryl sulfate, and mixtures thereof.

Exemplary natural oils include almond, apricot kernel, avocado, babassu,bergamot, black current seed, borage, cade, camomile, canola, caraway,carnauba, castor, cinnamon, cocoa butter, coconut, cod liver, coffee,corn, cotton seed, emu, eucalyptus, evening primrose, fish, flaxseed,geraniol, gourd, grape seed, hazel nut, hyssop, isopropyl myristate,jojoba, kukui nut, lavandin, lavender, lemon, litsea cubeba, macademianut, mallow, mango seed, meadowfoam seed, mink, nutmeg, olive, orange,orange roughy, palm, palm kernel, peach kernel, peanut, poppy seed,pumpkin seed, rapeseed, rice bran, rosemary, safflower, sandalwood,sasquana, savoury, sea buckthorn, sesame, shea butter, silicone,soybean, sunflower, tea tree, thistle, tsubaki, vetiver, walnut, andwheat germ oils. Exemplary synthetic oils include, but are not limitedto, butyl stearate, caprylic triglyceride, capric triglyceride,cyclomethicone, diethyl sebacate, dimethicone 360, isopropyl myristate,mineral oil, octyldodecanol, oleyl alcohol, silicone oil, and mixturesthereof.

In addition to the active ingredients, the liquid dosage forms maycomprise inert diluents commonly used in the art such as, for example,water or other solvents, solubilizing agents and emulsifiers such asethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzylalcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol,dimethylformamide, oils (e.g., cottonseed, groundnut, corn, germ, olive,castor, and sesame oils), glycerol, tetrahydrofurfuryl alcohol,polyethylene glycols and fatty acid esters of sorbitan, and mixturesthereof.

The composition may comprise water or other solvents, solubilizingagents and emulsifiers such as ethyl alcohol, isopropyl alcohol, ethylcarbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propyleneglycol, 1,3-butylene glycol, dimethylformamide, oils (e.g., cottonseed,groundnut, corn, germ, olive, castor, and sesame oils), glycerol,tetrahydrofurfuryl alcohol, polyethylene glycols and fatty acid estersof sorbitan, and mixtures thereof.

Formulations suitable for administration (e.g., to the ear canal)include, but are not limited to, liquid and/or semi-liquid preparationssuch as liniments, lotions, oil-in-water, and/or water-in-oil emulsionssuch as creams, ointments, and/or pastes, and/or solutions and/orsuspensions. Topically administrable formulations may, for example,comprise from about 1% to about 10% (w/w) therapeutic agent, althoughthe concentration of the therapeutic agent can be as high as thesolubility limit of the active ingredient in the solvent.

Polysaccharide Matrix Forming Agents

Also provided herein are compositions comprising a polysaccharide basedmatrix forming agent. The matrix forming agent may form a hydrogel bycrosslinking of a first polysaccharide derivative and a secondpolysaccharide derivative, wherein the first and second polysaccharidesare different. For example, the first polysaccharide may be anhyaluronic acid (HA) derivative comprising a first cross-linkablefunctional group, and the second polysaccharide may be a cellulosederivative comprising a second cross-linkable functional group. To givebut another example, the first polysaccharide is an HA derivativecomprising a first cross-linkable functional group, and the secondpolysaccharide is a dextran derivative comprising a secondcross-linkable functional group. In certain embodiments, the first andsecond cross-linkable functional groups are selected from the groupconsisting of amines, amides, aldehydes, esters, ketones, hydroxyls,hydrazines, hydrazides, maleimides, or sulfhydryls. In some embodiments,the first functional group is an amine, and the second functional groupis an aldehyde. In some embodiments, the first functional group is anamine, and the second functional group is a ketone. In some embodiments,the first functional group is an amine, hydroxyl, or sulfhydryl, and thesecond functional group is an ester. In some embodiments, the firstfunctional group is a maleimide, and the second functional group is asulfhydryl. In some embodiments, the first functional group is ahydrazine or hydrazide, and the second functional group is an aldehydeor ketone. In some embodiments, the first functional group is ahydrazide, and the second functional group is an aldehyde.

The hydrogels may formed by crosslinking a first polysaccharidederivative and a second polysaccharide derivative, wherein the first andsecond polysaccharides are the same and wherein the first polysaccharidederivative comprises a first cross-linkable functional group, and thesecond polysaccharide derivative comprises a second cross-linkablefunctional group, wherein the first and second cross-linkable functionalgroups are capable of crosslinking to one another. The polysaccharidemay be, e.g., glycosaminoglycan, HA, cellulose, dextran, or a derivativeof either.

HA, also referred to as hyaluronan or hyaluronate, is an unbranchedpolysaccharide containing repeating disaccharide subunits composed ofN-acetyl-D glucosamine and D-glucuronic acid. (See Laurent, T. C. (Ed).,Chemistry, Biology and Medical Applications of Hyaluronan and ItsDerivatives, London: Portland Press, 1998). HA also refers to any of itssalts, e.g., sodium hyaluronate, potassium hyaluronate, magnesiumhyaluronate, calcium hyaluronate, etc. The term “HA derivative” refersto HA that has been chemically modified from the native form.

Cellulose is a linear polymer of β-D-glucopyranose units joined to oneanother (Kamide, Cellulose And Cellulose Derivatives: MolecularCharacterization and Its Applications, Elsevier, 2005). The term“cellulose derivative” refers to cellulose that has been chemicallymodified from this native form. In certain embodiments, thepolysaccharide is a cellulose derivative such as methylcellulose (MC),carboxymethylcellulose (CMC), hydroxymethylcellulose (HMC),hydroxypropylcellulose (HPC), hydroxyethyl cellulose (HEC), orhydroxypropyl methylcellulose (HPMC), in which one or more of the OHgroups is replaced by OR, wherein R represents any of a variety ofmoieties.

Dextran is a complex, branched polysaccharide. Dextran includes manyglucose moieties joined together via α1→6 glycosidic linkages to formstraight chains. Branches typically begin from α1→3 linkages, but theymay also begin from α1→2 or α1→4 linkages. The term “dextran derivative”refers to dextran that has been chemically modified from this nativeform. In certain embodiments, the polysaccharide is a dextranderivative, in which one or more of the OH groups is replaced by OR,wherein R represents any of a variety of moieties.

Modifications to the polysaccharides may include the addition orcreation of new functional groups (e.g., amine, amide, aldehyde, ester,ketone, hydroxy, hydrazine, hydrazide, maleimide, sulfhydryl, etc.), inwhich case the polysaccharide is said to be “functionalized.” Theproportion of sugar subunits that are modified can vary, and the degreeof modification can be selected in order to control properties such asgelation time, half-life, stiffness, etc. Certain modifications retainthe native sugar backbone structure while other modifications open atleast some of the sugar rings. In some embodiments, the polysaccharidederivative is an aldehyde-containing derivative in which polysaccharidehas been treated with periodate.

It will be appreciated that in any of the polysaccharide derivatives,only a fraction of the sugar moieties in the polysaccharide aremodified. The extent of modification can vary. For example, in certainembodiments, between 5% and 99-100% of the relevant sugar moieties(e.g., glucuronic acid moieties in the case of the HA) are modified. Incertain embodiments, between 10% and 75% of the relevant sugar moietiesare modified. The extent of modification can be controlled by a varietyof methods. For example, the temperature, pH, and time during which thereaction is allowed to proceed can be varied, as can the concentrationof the reagents (e.g., carbodiimide, amide, dihydrazide, etc.). Toachieve a high degree of modification an excess of the modifyingagent(s), e.g., dihydrazide and/or carboiimide, may be used. Forexample, in one embodiment a 10-100 fold excess of dihydradize is addedto a solution comprising HA, and/or a 2-100 fold excess of carbodiimidereagent is then added to the reaction mixture. In certain embodiments,values for these parameters are selected so as to achieve a relativelyhigh degree of modification, e.g., between 50% and 99-100% of therelevant sugar moieties are modified. For example, between 50% and 80%of the relevant sugar moieties may be modified. However, the degree ofmodification is kept low enough so that the solution will remain in asuitably fluid state rather than becoming too viscous for easymanipulation and syringibility. In certain embodiments, between 10% and30%, or between 30% and 50% of the relevant sugar moieties are modified.

A variety of polysaccharide derivatives may be used. In certainembodiments, at least one of the polysaccharide derivatives is aderivative of HA. In certain embodiments, both of the polysaccharidederivatives are derivatives of HA. In certain embodiments, the matrixforming agent is separated into a first and second polysaccharidederivative which form a matrix or gel upon mixing. In some embodiments,the first polysaccharide derivative comprises a first type ofcross-linkable functional group, and the second polysaccharidederivative comprises a second type of cross-linkable functional group,wherein the two types of cross-linkable functional groups formcross-links between the two polysaccharide derivative upon mixing. Thepolysaccharide derivatives may be kept separate as two components of thecompositions, which are mixed during administration, e.g., duringapplication to the ear canal with a double barrel syringe. In certainembodiments, the polysaccharide is one that is not specifically degradedby an enzyme endogenous to human beings.

In certain embodiments, at least one of the polysaccharide derivativesis a derivative of cellulose. For example, in certain embodiments, thefirst polysaccharide derivative is a derivative of HA, and the secondpolysaccharide derivative is a derivative of cellulose.

In certain embodiments, at least one of the polysaccharide derivativesis a derivative of dextran. For example, in certain embodiments, thefirst polysaccharide derivative is a derivative of HA, and the secondpolysaccharide derivative is a derivative of dextran.

The first and second polysaccharide derivatives comprise first andsecond functional groups, respectively, that react with one another toform covalent bonds that join the first and second polysaccharidederivatives to one another. The solutions are thus applied as liquidsand are contacted with one another and optionally mixed together eitherimmediately before or at the time of administration or contact oneanother following administration. Formation of a sufficient number ofcrosslinks causes a transition from a liquid to a semi-solid or gel-likestate.

In certain embodiments, a polysaccharide derivative comprising at leasttwo different cross-linkable functional groups is employed, wherein thecross-linkable functional groups react with one another to formcrosslinks under physiological conditions. The cross-linkable functionalgroups may be selected so that they substantially do not react with oneanother until exposed to physiological conditions of pH, temperature,and/or salt concentration. Thus it will be appreciated that matrixforming agent does not require two distinguishable HA derivatives butmay instead employ a single species that comprises multiple differentfunctional groups capable of becoming crosslinked.

A variety of different polysaccharide derivatives (e.g., HA derivatives)are of use in the composition. In certain embodiments, the first andsecond cross-linkable functional groups react in sufficient amounts andwith sufficient rapidity so as to allow hydrogel formation within a timeframe following contact of the solutions with one another. In certainembodiments, the hydrogel forms within between 1-3 seconds and 5minutes, between 1-3 seconds and 3 minutes, between 1-3 seconds and 60seconds, between 1-3 seconds and 30 seconds, or between 1-3 seconds and15 seconds, following contact of the solutions with one another, e.g.,following administration. Typically the solutions are mixed togethereither immediately before or concurrently with their administration to asite within the body (e.g., ear canal). For example, the solutions maybe administered using a multiple barrel injection device, e.g., amultiple barrel syringe, wherein each solution is contained in aseparate receptacle or barrel prior to administration. The solutions maycontact each other during the administration process and/or thereafter.Preferably the derivatives become crosslinked under physiologicalconditions, e.g., in an aqueous environment at a pH between 6.0 and 8.0.

A variety of cross-linkable polysaccharide derivatives and methods forforming them may be employed. In certain embodiments, the polysaccharidederivatives become crosslinked to one another without needing a separatecrosslinking agent, e.g., the first and second derivatives comprisefunctional groups that react with one another to form a covalent bond.In certain embodiments, the polysaccharide derivatives react with oneanother to produce a nontoxic, biocompatible product, e.g., water. Incertain embodiments, neither of the polysaccharide derivatives ismodified by using a crosslinking agent. In certain embodiments, thepolysaccharide derivatives become crosslinked without requiring light.

It will be appreciated that in any of the above embodiments, only afraction of the available functional groups on the first and secondpolysaccharide derivatives will become crosslinked. The crosslinkingdensity can be controlled, e.g., by appropriately selecting themolecular weights of the polysaccharide derivatives. Exemplarycrosslinking densities range from about 1×10⁶ to about 1×10⁸ mol/cm³. Incertain embodiments, the crosslinking density ranges from 3-50×10⁷mol/cm³.

Polysaccharide derivatives functionalized as described above can becrosslinked by allowing derivatives comprising different functionalgroups to react with one another. For example, (i) a firstpolysaccharide derivative comprising an aldehyde can react with a secondpolysaccharide derivative comprising an amine; (ii) a firstpolysaccharide derivative comprising an active ester such as an NHSester can react with a second polysaccharide derivative comprising anamine; (iv) a first polysaccharide derivative comprising a hydrazide canreact with a second HA derivative comprising an aldehyde, etc. In oneembodiment of particular interest the first solution contains apolysaccharide derivative functionalized with a dihydrazide, and thesecond solution contains polysaccharide oxidized to form aldehyde groups(See, e.g., FIG. 8).

In certain embodiments, the composition comprises a matrix forming agentcomprising a polysaccharide derivative in solution, wherein theconcentration of the polysaccharide derivative is greater than 5 mg/ml,e.g., up to 150 mg/ml. In certain embodiments, the concentration of thepolysaccharide derivative is greater than 10 mg/ml. In otherembodiments, the concentration of the polysaccharide derivative isgreater than 15 mg/ml, greater than 20 mg/ml, or greater than 25 mg/ml.Herein, a polysaccharide derivative greater than 25 mg/ml is referred toas a “high concentration.” The solution preferably has a sufficientlylow viscosity such that it can be readily manipulated, e.g., so thateasy syringibility exists.

In certain embodiments, at least one of the polysaccharide derivativessuitable for in situ crosslinking to form a gel, comprises a portionthat comprises a non-polysaccharide polymer, e.g., the polysaccharidederivative comprises a polysaccharide or derivative thereof covalentlyattached to one or more non-polysaccharide polymers. Non-polysaccharidemeans that the polymer contains less than 1% sugar monomers by weight,number, or both, e.g., the polymer contains essentially no sugars. Incertain embodiments, the non-polysaccharide portion comprises between 1%and 10%-90% of the polymer by weight and/or between 1% and 10%-90% ofthe monomers are non-sugar monomers. The attachment may occur at anyposition in the polysaccharide chain, e.g., either of the ends of thechain or at one of the internally located sugar moieties resulting ineither a linear or branched structure. The non-polysaccharide polymercan be any of a variety of polymers, e.g., any non-polysaccharidepolymer capable of serving as a hydrogel precursor when covalentlyattached to a polysaccharide derivative.

In certain embodiments, the matrix forming agent comprises first andsecond crosslinkable hydrogel precursors, wherein one of the hydrogelprecursors comprises or consists of a polysaccharide derivative such asan HA, cellulose, or dextran derivative and the other hydrogel precursorcomprises or consists of a non-polysaccharide polymer (i.e., less than1% of the polymer by weight, or less than 1% of the monomers aresugars). Exemplary non-polysaccharide polymers capable of becomingcrosslinked to a polysaccharide derivative to form a hydrogel includebut are not limited to polyethers such as polyethylene glycol (PEG) orpolypropylene glycol (PPG), polyethylene oxides (PEO), polyvinyl alcohol(PVA), polyvinyl pyrrolidone (PVP), polypeptides such as gelatin,chitosan, or poly(1-glutamic acid), and derivatives of any of these, orconjugates, blends, or composites comprising any of these.

While polysaccharide derivatives are described in detail, in yet otherembodiments, the hydrogel is formed by crosslinking twonon-polysaccharide polymers in situ. in a hydrogel that inhibitsadhesions. Each of the non-polysaccharide polymers comprises afunctional group, wherein the functional groups are capable of reactingwith one another to form covalent bonds. Suitable functional groups arethose described above for crosslinking of polysaccharide derivatives.Exemplary non-polysaccharide polymers include those described above thatcontain or may be modified to contain suitable functional groups forcrosslinking.

Matrix Forming Agents (and Compositions Thereof)

In one aspect, provided herein are matrix forming agents describedherein. In certain embodiments, the matrix forming agent comprises apolymer. In certain embodiments, the matrix forming agent comprisespolymers that gel via electrostatic interactions. In certainembodiments, the matrix forming agent comprises polymers that displayshear thinning. In certain embodiments, the matrix forming agentcomprises rheological blends of polymers. In certain embodiments,rheological polymer blends comprise two different polymers wherein theviscoelastic properties of the rheological polymer blends are moregel-like than those of the constituent polymers measured individuallyThe polymer may be a block copolymer. In certain embodiments, thepolymer is not a block copolymer. In certain embodiments, the polymer isa thermosensitive polymer. In certain embodiments, the polymer ispoly(N-isopropylacrylamide) (NIPAAm). In certain embodiments, the matrixforming agent or combination of matrix forming agents is not modified bya polyphosphoester. In certain embodiments, the matrix forming agent orcombination of matrix forming agents is modified by a polyphosphoester.In certain embodiments, the matrix forming agent or combination ofmatrix forming agents comprises a polymer of Formula:

wherein each occurrence of Y is independently —R¹ or -L²R²;

-   -   each occurrence of R¹ is independently hydrogen, optionally        substituted alkyl, optionally substituted alkenyl, optionally        substituted alkynyl, optionally substituted aryl, or optionally        substituted heteroaryl;    -   each occurrence of L² is independently a bond, optionally        substituted alkylene, optionally substituted alkenylene,        optionally substituted alkynylene, optionally substituted        heteroalkylene, optionally substituted heteroalkenylene, or        optionally substituted heteroalkynylene;    -   each occurrence of R² is independently optionally substituted        acyl, optionally substituted carbocyclyl, optionally substituted        heterocyclyl, optionally substituted aryl, optionally        substituted heteroaryl, —OR^(b), —N(R^(b))₂, or an oxygen        protecting group;    -   each occurrence of R^(3A) is independently hydrogen, optionally        substituted alkyl, optionally substituted alkenyl, optionally        substituted alkynyl, optionally substituted aryl, optionally        substituted heteryaryl, optionally substituted acyl, —OR^(b), or        —N(R^(b))₂;    -   each occurrence of R^(b) is independently optionally substituted        alkyl, optionally substituted alkenyl, optionally substituted        alkynyl, optionally substituted carbocyclyl, optionally        substituted heterocyclyl, optionally substituted aryl,        optionally substituted heteroaryl, optionally substituted acyl,        an oxygen protecting group, or a nitrogen protecting group, or        two R^(b) taken together with the nitrogen to which they are        attached form an optionally substituted heterocyclic or        optionally substituted heteroaryl ring;    -   each of G^(1A) and G^(2A) is independently hydrogen, halogen,        optionally substituted amine, optionally substituted alkyl,        optionally substituted aryl, or optionally substituted        heteroaryl, optionally substituted acyl, optionally substituted        phosphate, or an oxygen protecting group; and

each of p, q, r, s, and t is independently an integer between 0 and 200,inclusive, wherein the sum of p and t is at least 1, and the sum of q,r, and s is at least 1.

In certain embodiments, the matrix forming agent or combination ofmatrix forming agents comprises a polymer of Formula:

-   -   wherein:    -   each occurrence of Z is independently —R⁴;    -   each occurrence of R⁴ is independently optionally substituted        alkyl;    -   each of G^(1A) and G^(2A) is independently hydrogen, optionally        substituted alkyl, optionally substituted aryl, or optionally        substituted heteroaryl, optionally substituted acyl, optionally        substituted phosphate, or an oxygen protecting group; and    -   each of p, q, r, s, and t is independently an integer between 0        and 200, wherein the sum of p and t is at least 1, and the sum        of q, r, and s is at least 1;        -   the composition forms a gel at temperatures above a phase            transition temperature; and the phase transition temperature            is less than about 37° C.;    -   and at least one of conditions (i), (ii), and (iii) are met:    -   (i) the phase transition temperature of the composition is less        than the phase transition temperature of a reference composition        plus about 5° C.;    -   (ii) the storage modulus of the composition is greater than        about 15% of the storage modulus of the reference composition at        a temperature of about 37° C.; and    -   (iii) the loss modulus of the composition is between about 80%        and about 120% of the loss modulus of the reference composition        at a temperature of about 37° C.;        wherein the reference composition is the composition in the        absence of the permeation enhancer or combination of permeation        enhancers.

In some embodiments, the matrix forming agent comprises a poloxamer.Exemplary poloxamers include, but are not limited to: poloxamer 407,poloxamer 188, poloxalene, poloxamer 124, poloxamer 237, or poloxamer338, Pluronic® 10R5, Pluronic® 17R2, Pluronic® 17R4, Pluronic® 25R2,Pluronic® 25R4, Pluronic® 31R1, Pluronic® F 108 Cast Solid Surfactant,Pluronic® F 108 NF, Pluronic® F 108 Pastille, Pluronic® F 108NF PrillPoloxamer 338, Pluronic® F 127 NF, Pluronic® F 127 NF 500 BHT Prill,Pluronic® F 127 NF Prill Poloxamer 407, Pluronic® F 38, Pluronic® F 38Pastille, Pluronic® F 68, Pluronic® F 68 LF Pastille, Pluronic® F 68 NF,Pluronic® F 68 NF Prill Poloxamer 188, Pluronic® F 68 Pastille,Pluronic® F 77, Pluronic® F 77 Micropastille, Pluronic® F 87, Pluronic®F 87 NF, Pluronic® F 87 NF Prill Poloxamer 237, Pluronic® F 88,Pluronic® F 88 Pastille, Pluronic® FT L 61, Pluronic® L 10, Pluronic® L101, Pluronic® L 121, Pluronic® L 31, Pluronic® L 35, Pluronic® L 43,Pluronic® L 61, Pluronic® L 62, Pluronic® L 62 LF, Pluronic® L 62D,Pluronic® L 64, Pluronic® L 81, Pluronic® L 92, Pluronic® L44 NF INHsurfactant Poloxamer 124, Pluronic® N 3, Pluronic® P 103, Pluronic® P104, Pluronic® P 105, Pluronic® P 123 Surfactant, Pluronic® P 65,Pluronic® P 84, Pluronic® P 85, Synperonic® PE/F 108, Synperonic®PE/P105, Synperonic® PE/P84, Synperonic®, Synperonic® PE/L31,Synperonic® PE/L61, Synperonic® PE/L101, Synperonic® PE/L121,Synperonic® PE/L42, Synperonic® PE/L62, Synperonic® PE/L92, Synperonic®PE/L44, Synperonic® PE/L64, Synperonic® PE/P84, Synperonic® PE/P75,Synperonic® PE/P103, Synperonic® PE/F87, Synperonic® PE/F127,Synperonic® PE/F38, Synperonic® PE/F68, Kolliphor® P 188, Kolliphor® P407, Kolliphor® P 188 micro, Kolliphor® P 407 micro, Kolliphor® P237,Kolliphor® P 338, Kolliphor® EL, Kolliphor® HS 15, Kolliphor® PS 80,Kolliphor® PS 60, Kolliphor® RH 40, Kolliphor® TPG S, Kolliphor® CS L,Kolliphor® CS A, Kolliphor® CS S, Kolliphor® CS B, Kolliphor® CS 20, andKolliphor® CS 12. In some embodiments, the matrix forming agentcomprises any of the foregoing poloxamers, a derivative thereof, or ablock copolymer thereof.

In certain embodiments, the matrix forming agent comprises poloxamer407, poloxamer 188, poloxalene, poloxamer 124, poloxamer 237, orpoloxamer 338. In certain embodiments, the block copolymer comprisespoloxamer 407. In certain embodiments, the matrix forming comprises apoloxamer with phosphoester monomers. In certain embodiments, the matrixforming comprises poloxamer 407 with phosphoester monomers. In certainembodiments, the matrix forming agent or combination of matrix formingagents comprises a polymer of Formula:

-   -   wherein:    -   each occurrence of Z is independently —R⁴;    -   each occurrence of R⁴ is independently optionally substituted        alkyl;    -   each of G^(1A) and G^(2A) is independently hydrogen, optionally        substituted alkyl, optionally substituted aryl, or optionally        substituted heteroaryl, optionally substituted acyl, optionally        substituted phosphate, or an oxygen protecting group; and each        of p and t is independently an integer between 0 and 200,        inclusive, wherein the sum of p and t is at least 1;        -   the composition forms a gel at temperatures above a phase            transition temperature; and the phase transition temperature            is less than about 37° C.;    -   and at least one of conditions (i), (ii), and (iii) are met:    -   (i) the phase transition temperature of the composition is less        than the phase transition temperature of a reference composition        plus about 5° C.;    -   (ii) the storage modulus of the composition is greater than        about 15% of the storage modulus of the reference composition at        a temperature of about 37° C.; and    -   (iii) the loss modulus of the composition is between about 80%        and about 120% of the loss modulus of the reference composition        at a temperature of about 37° C.; wherein the reference        composition is the composition in the absence of the permeation        enhancer or combination of permeation enhancers.

Polymers of Formula (I′) comprise R^(3A). In certain embodiments, eachR^(3A) is the same substituent. In certain embodiments, each R^(3A) in apolymer is one of two specific substituents. In certain embodiments,each R^(3A) in a polymer is one of three specific substituents. Incertain embodiments, each R^(3A) in a polymer is one of four specificsubstituents. In certain embodiments, each R^(3A) in a polymer is one offive specific substituents. In certain embodiments, each R^(3A) in apolymer is one of six specific substituents. In certain embodiments,each R^(3A) in a polymer is one of seven or more specific substituents.

As generally described herein each occurrence of R^(3A) is independentlyoptionally hydrogen, substituted alkyl, optionally substituted alkenyl,optionally substituted alkynyl, optionally substituted aryl, optionallysubstituted heteryaryl, optionally substituted acyl, —OR^(b), or—N(R^(b))₂. In certain embodiments, each occurrence of R^(3A) isindependently unsubstituted alkyl.

In certain embodiments, R^(3A) is hydrogen. In certain embodiments,R^(3A) is optionally substituted alkyl, e.g., optionally substitutedC₁₋₆ alkyl, optionally substituted C₁₋₂ alkyl, optionally substitutedC₂₋₃ alkyl, optionally substituted C₃₋₄ alkyl, optionally substitutedC₄₋₅ alkyl, or optionally substituted C₅₋₆ alkyl. In certainembodiments, R^(3A) is unsubstituted alkyl, e.g., unsubstituted C₁₋₆alkyl, unsubstituted C₁₋₂ alkyl, unsubstituted C₂₋₃ alkyl, unsubstitutedC₃₋₄ alkyl, unsubstituted C₄₋₅ alkyl, or unsubstituted C₅₋₆ alkyl. Incertain embodiments, R^(3A) is unsubstituted C₁₋₂₀ alkyl. In certainembodiments, R^(3A) is unsubstituted C₁₋₁₂ alkyl. In certainembodiments, R^(3A) is methyl. In certain embodiments, R^(3A) is ethyl,propyl, or butyl. In certain embodiments, R^(3A) is haloalkyl, e.g.,—CHF₂, —CHCl₂, —CH₂CHF₂, —CH₂CHCl₂. In certain embodiments, R^(3A) isperhaloalkyl, e.g., —CF₃, —CF₂CF₃, —CCl₃. In certain embodiments, R^(3A)is hydroxyalkyl, e.g., —CH₂OH, —CH₂CH₂OH, —CH₂OR^(b), —CH₂CH₂OR^(b). Incertain embodiments, R^(3A) is aminoalkyl, e.g., —CH₂NH₂, —CH₂CH₂NH₂,—CH₂NMe₂, —CH₂CH₂NMe₂, —CH₂N(R^(b))₂, —CH2CH2N(R^(b))₂.

In certain embodiments, R^(3A) is optionally substituted alkenyl, e.g.,optionally substituted C₂₋₆ alkenyl. In certain embodiments, R^(3A) isunsubstituted alkenyl, e.g., unsubstituted C₂₋₆ alkenyl. In certainembodiments, R^(3A) is vinyl, allyl, or prenyl. In certain embodiments,R^(3A) is optionally substituted alkynyl, e.g., optionally substitutedC₂₋₆ alkynyl. In certain embodiments, R^(3A) is unsubstituted alkynyl,e.g., unsubstituted C₂₋₆ alkynyl.

In certain embodiments, R^(3A) is optionally substituted aryl, e.g.,optionally substituted phenyl. In certain embodiments, R^(3A) isunsubstituted aryl, e.g., unsubstituted phenyl. In certain embodiments,R^(3A) is optionally substituted heteroaryl, e.g., optionallysubstituted 5-6 membered heteroaryl, or optionally substituted 9-10membered bicyclic heteroaryl. In certain embodiments, R^(3A) isunsubstituted heteroaryl, e.g., unsubstituted 5-6 membered heteroaryl,or unsubstituted 9-10 membered bicyclic heteroaryl.

In certain embodiments, R^(3A) is optionally substituted acyl, e.g.,—CHO, —CO₂H, or —C(═O)NH₂. In certain embodiments, R^(3A) is optionallysubstituted carbonyl. In certain embodiments, R^(3A) is —C(═O)R^(b),—C(═O)OR^(b), —C(═O)NH(R^(b)), or —C(═O)N(R^(b))₂. In certainembodiments, R^(3A) is —C(═O)R^(b), and R^(b) is optionally substitutedalkyl, e.g., —C(═O)Me. In certain embodiments, R³ is —C(═O)R^(b), andR^(b) is optionally substituted alkenyl. In certain embodiments, R^(3A)is —C(═O)R^(b), and R^(b) is optionally substituted carbocyclyl,heterocyclyl, aryl, or heteroaryl. In certain embodiments, R^(3A) is—C(═O)OR^(b), and R^(b) is optionally substituted alkyl. In certainembodiments, R^(3A) is —C(═O)OR^(b), and R^(b) is optionally substitutedalkenyl. In certain embodiments, R^(3A) is —C(═O)OR^(b), and R^(b) isoptionally substituted carbocyclyl, heterocyclyl, aryl, or heteroaryl.In certain embodiments, R^(3A) is —C(═O)N(R^(b))₂, and at least oneR^(b) is optionally substituted alkyl. In certain embodiments, R³ is—C(═O)NHR^(b), and R^(b) is optionally substituted alkyl. In certainembodiments, R^(3A) is —C(═O)NHR^(b), and R^(b) is optionallysubstituted alkenyl. In certain embodiments, R^(3A) is —C(═O)NHR^(b),and R^(b) is optionally substituted carbocyclyl, heterocyclyl, aryl, orheteroaryl. In certain embodiments, R^(3A) is optionally substitutedvinylcarbonyl (e.g., —C(═O)CH═CH₂, —C(═O)CMe=CH₂).

In certain embodiments, R^(3A) is —OR^(b), e.g., —OH. In certainembodiments, R^(3A) is —OR^(b), and R^(b) is optionally substitutedalkyl. In certain embodiments, R^(3A) is —OR^(b), and R^(b) isunsubstituted C₁₋₆ alkyl. In certain embodiments, R^(3A) is —OR^(b), andR^(b) is optionally substituted alkenyl. In certain embodiments, R^(3A)is —OR^(b), and R^(b) is optionally substituted carbocyclyl, optionallysubstituted heterocyclyl, optionally substituted aryl optionallysubstituted heteroaryl. In certain embodiments, R^(3A) is —OR^(b), andR^(b) is unsubstituted carbocyclyl, unsubstituted heterocyclyl,unsubstituted aryl unsubstituted heteroaryl. In certain embodiments,R^(3A) is —OR^(b), and R^(b) is optionally substituted acyl, e.g.,R^(3A) is —OC(═O)R^(b), —OC(═O)OR^(b), or —OC(═O)N(R^(b))₂. In certainembodiments, R^(3A) is —OR^(b), and R^(b) is an oxygen protecting group(e.g., silyl, TBDPS, TBDMS, TIPS, TES, TMS, MOM, THP, t-Bu, Bn, allyl,acetyl, pivaloyl, benzoyl).

In certain embodiments, R^(3A) is —N(R^(b))₂, e.g., —NH₂, —NHR^(b). Incertain embodiments, R^(3A) is —NH(R^(b)), and R^(b) is optionallysubstituted alkyl. In certain embodiments, R³ is —N(R^(b))₂, and atleast one R^(b) is optionally substituted alkyl. In certain embodiments,R^(3A) is —NH(R^(b)), and R^(b) is unsubstituted alkyl. In certainembodiments, R^(3A) is —N(R^(b))₂, and at least one R^(b) isunsubstituted alkyl. In certain embodiments, R^(3A) is —NHR^(b), andR^(b) is optionally substituted carbocyclyl, optionally substitutedheterocyclyl, optionally substituted aryl, or optionally substitutedheteroaryl. In certain embodiments, R^(3A) is —NHR^(b), and R^(b) isunsubstituted carbocyclyl, unsubstituted heterocyclyl, unsubstitutedaryl, or unsubstituted heteroaryl. In certain embodiments, R^(3A) is—NHR^(b), and R^(b) is optionally substituted acyl, e.g., R^(3A) is—NHC(═O)R^(b), —NHC(═O)OR^(b), or —NHC(═O)NHR^(b). In certainembodiments, R^(3A) is —N(R^(b))₂, and at least one R^(b) is a nitrogenprotecting group (e.g., Bn, Boc, Cbz, Fmoc, trifluoroacetyl,triphenylmethyl, acetyl, Ts). In certain embodiments, R^(3A) is—N(R^(b))₂, and both R^(b) are joined to form an optionally substitutedheterocyclic or optionally substituted heteroaryl ring. In certainembodiments, R^(3A) is —N(R^(b))₂, and both R^(b) are joined to form anunsubstituted heterocyclic or unsubstituted heteroaryl ring.

In some embodiments, G^(1A) and G^(2A) are the same. In someembodiments, G^(1A) and G^(2A) are different. In certain embodiments,G^(1A) is hydrogen. In certain embodiments, G^(1A) is optionallysubstituted alkyl. In certain embodiments, G^(1A) is optionallysubstituted acyl. In certain embodiments, G^(1A) is optionallysubstituted optionally substituted phosphate (e.g., —P(═O)(OH)₂,—P(═O)(O-alkyl)₂, —P(═O)(OH)(O-alkyl), —P(═O)(OH)(O—Y),—P(═O)(O-alkyl)(O—Y)). In certain embodiments, G^(1A) is an oxygenprotecting group (e.g., silyl, TBDPS, TBDMS, TIPS, TES, TMS, MOM, THP,t-Bu, Bn, allyl, acetyl, pivaloyl, benzoyl). In certain embodiments,G^(1A) is hydrogen. In certain embodiments, G^(1A) is optionallysubstituted alkyl. In certain embodiments, G^(1A) is optionallysubstituted acyl. In certain embodiments, G^(1A) is optionallysubstituted optionally substituted phosphate (e.g., —P(═O)(OH)₂,—P(═O)(O-alkyl)₂, —P(═O)(OH)(O-alkyl), —P(═O)(OH)(O—Y),—P(═O)(O-alkyl)(O—Y)). In certain embodiments, G^(1A) is an oxygenprotecting group (e.g., silyl, TBDPS, TBDMS, TIPS, TES, TMS, MOM, THP,t-Bu, Bn, allyl, acetyl, pivaloyl, benzoyl). In certain embodiments,G^(1A) is optionally substituted aryl, e.g., optionally substitutedphenyl. In certain embodiments, G^(1A) is unsubstituted aryl, e.g.,unsubstituted phenyl. In certain embodiments, G^(1A) is optionallysubstituted heteroaryl, e.g., optionally substituted 5-6 memberedheteroaryl, or optionally substituted 9-10 membered bicyclic heteroaryl.In certain embodiments, G^(1A) is unsubstituted heteroaryl, e.g.,unsubstituted 5-6 membered heteroaryl, or unsubstituted 9-10 memberedbicyclic heteroaryl.

In certain embodiments, G^(2A) is hydrogen. In certain embodiments,G^(2A) is optionally substituted alkyl. In certain embodiments, G^(2A)is optionally substituted acyl. In certain embodiments, G^(2A) isoptionally substituted optionally substituted phosphate (e.g.,—P(═O)(OH)₂, —P(═O)(O-alkyl)₂, —P(═O)(OH)(O-alkyl), —P(═O)(OH)(O—Y),—P(═O)(O-alkyl)(O—Y)). In certain embodiments, G^(2A) is an oxygenprotecting group (e.g., silyl, TBDPS, TBDMS, TIPS, TES, TMS, MOM, THP,t-Bu, Bn, allyl, acetyl, pivaloyl, benzoyl). In certain embodiments, G²is hydrogen. In certain embodiments, G^(2A) is optionally substitutedalkyl. In certain embodiments, G² is optionally substituted acyl. Incertain embodiments, G^(2A) is optionally substituted optionallysubstituted phosphate (e.g., —P(═O)(OH)₂, —P(═O)(O-alkyl)₂,—P(═O)(OH)(O-alkyl), —P(═O)(OH)(O—Y), —P(═O)(O-alkyl)(O—Y)). In certainembodiments, G^(2A) is an oxygen protecting group (e.g., silyl, TBDPS,TBDMS, TIPS, TES, TMS, MOM, THP, t-Bu, Bn, allyl, acetyl, pivaloyl,benzoyl). In certain embodiments, G^(2A) is optionally substituted aryl,e.g., optionally substituted phenyl. In certain embodiments, G^(2A) isunsubstituted aryl, e.g., unsubstituted phenyl. In certain embodiments,G^(2A) is optionally substituted heteroaryl, e.g., optionallysubstituted 5-6 membered heteroaryl, or optionally substituted 9-10membered bicyclic heteroaryl. In certain embodiments, G^(2A) isunsubstituted heteroaryl, e.g., unsubstituted 5-6 membered heteroaryl,or unsubstituted 9-10 membered bicyclic heteroaryl. In certainembodiments, G^(1A) and G^(2A) are both hydrogen.

In certain embodiments, each occurrence of Z is independently —R⁴. Asgenerally described herein each occurrence of R⁴ is independentlyoptionally substituted alkyl. In certain embodiments, R⁴ is optionallysubstituted alkyl, e.g., optionally substituted C₁₋₆ alkyl, optionallysubstituted C₁₋₂ alkyl, optionally substituted C₂₋₃ alkyl, optionallysubstituted C₃₋₄ alkyl, optionally substituted C₄₋₅ alkyl, or optionallysubstituted C₅₋₆ alkyl. In certain embodiments, R⁴ is unsubstitutedalkyl, e.g., unsubstituted C₁₋₆ alkyl, unsubstituted C₁₋₂ alkyl,unsubstituted C₂₋₃ alkyl, unsubstituted C₃₋₄ alkyl, unsubstituted C₄₋₅alkyl, or unsubstituted C₅₋₆ alkyl. In certain embodiments, R⁴ isunsubstituted C₁₋₂₀ alkyl. In certain embodiments, R⁴ is unsubstitutedC₁-12 alkyl. In certain embodiments, R⁴ is methyl. In certainembodiments, R⁴ is ethyl, propyl, or butyl. In certain embodiments, R⁴is ethyl. In certain embodiments, R⁴ is propyl. In certain embodiments,R⁴ is butyl. In certain embodiments, R⁴ is n-butyl. In certainembodiments, R⁴ is s-butyl. In certain embodiments, R⁴ is of formula:

wherein each occurrence of n is independently an integer between 0 and20, inclusive.

In certain embodiments, R⁴ is of formula:

wherein each occurrence of n is independently an integer between 0 and20, inclusive. In certain embodiments, R⁴ is of formula:

In certain embodiments, R⁴ is of formula:

In certain embodiments, R⁴ is of formula:

In certain embodiments, R⁴ is of formula:

In certain embodiments, p is 0. In certain embodiments, p is an integerbetween 1 and 100, inclusive. In some embodiments, p is an integerbetween 10 and 100, inclusive. In some embodiments, p is an integerbetween 10 and 50, inclusive. In some embodiments, p is an integerbetween 10 and 25, inclusive. In some embodiments, p is an integerbetween 1 and 10, inclusive. In certain embodiments, p is 5.

In certain embodiments, t is 0. In certain embodiments, t is an integerbetween 1 and 100, inclusive. In some embodiments, t is an integerbetween 10 and 100, inclusive. In some embodiments, t is an integerbetween 10 and 50, inclusive. In some embodiments, t is an integerbetween 10 and 25, inclusive. In some embodiments, t is an integerbetween 1 and 10, inclusive. In certain embodiments, t is 5. In certainembodiments, p is 5 and t is 5.

In certain embodiments, the polymer is of the formula:

In certain embodiments, the matrix forming agent or combination ofmatrix forming agents comprises a polymer of Formula (I′) is part of acomposition. In certain embodiments, the matrix forming agent orcombination of matrix forming agents comprises a polymer of Formula (IA)is part of a composition. In certain embodiments, the matrix formingagent or combination of matrix forming agents comprises a polymer ofFormula (II) is part of a composition.

Methods of Treatment and Uses

Methods of using the various embodiments of the compositions describedherein are generally directed to methods of treating an infectiousdisease or an ear disease. In certain embodiments, the compositionsdescribed herein are used in a method of treating an infectious disease.In certain embodiments, the matrix forming agents described herein areused in a method of treating an infectious disease. In certainembodiments, the compositions described herein are used in a method oftreating an ear disease. In certain embodiments, the compositionsdescribed herein are used in a method of treating an infectious eardisease. Methods of using the various embodiments of the compositionsdescribed herein are generally directed to methods of treating aninfectious disease. In various aspects, the compositions may be used todeliver therapeutic or diagnostic agents across the tympanic membrane.Therefore, the compositions are particularly useful in treating diseasesof the middle and/or inner ear. In certain embodiments, the compositionsdescribed herein are used in a method of treating diseases of the middleear. In certain embodiments, the compositions described herein are usedin a method of treating diseases of the inner ear.

In certain embodiments, the subject described herein is a human. Incertain embodiments, the subject is a non-human animal. In certainembodiments, the subject is a mammal. In certain embodiments, thesubject is a non-human mammal. In certain embodiments, the subject is adomesticated animal, such as a dog, cat, cow, pig, horse, sheep, orgoat. In certain embodiments, the subject is a companion animal, such asa dog or cat. In certain embodiments, the subject is a livestock animal,such as a cow, pig, horse, sheep, or goat. In certain embodiments, thesubject is a zoo animal. In another embodiment, the subject is aresearch animal, such as a rodent (e.g., mouse, rat), dog, pig, ornon-human primate.

In various aspects, compositions described herein can be used to treatear diseases, including, but not limited to, ear infections, developmentof fibroids in the middle ear, or otosclerosis. In certain embodiments,the matrix forming agents described herein can be used to treat eardiseases, including, but not limited to, ear infections, development offibroids in the middle ear, or otosclerosis. In various other aspects,compositions described herein may be used may treat vertigo, Meniere'sdisease, mastoiditis, cholesteatoma, labyrinthitis, perilymph fistula,superior canal dehiscence syndrome, otorrhea, otalgia, tinnitus,barotrauma, cancers of the ear, autoimmune inner ear disease acousticneuroma, benign paroxysmal positional vertigo, herpes zoster oticus,purulent labyrinthitis, vestibular neuronitis, eardrum perforation, ormyringitis. In various other aspects, compositions described herein maybe used may treat vertigo, Meniere's disease, mastoiditis,cholesteatoma, labyrinthitis, perilymph fistula, superior canaldehiscence syndrome, otorrhea, otalgia, tinnitus, barotrauma, cancers ofthe ear, autoimmune inner ear disease acoustic neuroma, benignparoxysmal positional vertigo, herpes zoster oticus, purulentlabyrinthitis, vestibular neuronitis, eardrum perforation, ormyringitis. In certain embodiments, the matrix forming agents describedherein may be used may treat vertigo, Meniere's disease, mastoiditis,cholesteatoma, labyrinthitis, perilymph fistula, superior canaldehiscence syndrome, otorrhea, otalgia, tinnitus, barotrauma, cancers ofthe ear, autoimmune inner ear disease acoustic neuroma, benignparoxysmal positional vertigo, herpes zoster oticus, purulentlabyrinthitis, vestibular neuronitis, eardrum perforation, ormyringitis. In some embodiments, the methods disclosed herein are usedfor treating otitis media (OM). Different forms of OM, which may betreated by the methods disclosed herein, may be differentiated by thepresence of fluid (effusion) and/or by the duration or persistence ofinflammation. In certain embodiments, the infectious disease is acuteotitis media, chronic otitis media, or secretory otitis media.Effusions, if present, can be of any consistency, from water-like(serous) to viscid and mucous-like (mucoid), to pus-like (purulent);duration is classified as acute, subacute, or chronic. OM with effusion(OME) indicates inflammation with middle ear fluid (MEF), but in theabsence of any indications of acute infection. Acute OM (AOM), with orwithout effusion, is characterized by rapid onset of the signs andsymptoms associated with acute infection in the middle ear (e.g.,otalgia, fever). In some embodiments, the methods are used for treatingotitis media associated with infection by any of a number of pathogenicbacteria, including, for example, Streptococcus pneumoniae, Haemophilusinfluenzae, and Moraxella catarrhalis.

The infectious disease may be a bacterial infection. In certainembodiments, the bacterial infection is a Streptococcus, Haemophilus, orMoraxella infection. In certain embodiments, the bacterial infection isa Staphylococcus, Escherichia, or Bacillus infection. In certainembodiments, the bacterial infection is an H. influenzae infection. Incertain embodiments, the bacterial infection is a S. pneumoniaeinfection. In certain embodiments, the bacterial infection is an M.catarrhalis infection. In certain embodiments, the infectious disease isan ear infection. In certain embodiments, the infectious disease isotitis media.

In various embodiments, administration of the inventive compositionsconsists of applying the composition into a subject's ear canal. Incertain embodiments, applying the composition into a subject's ear canalcomprises spraying the composition into a subject's ear canal. Incertain embodiments, administration of the inventive compositionsconsists of applying the composition into the inner ear of a subject. Incertain embodiments, administration of the inventive compositionsconsists of applying the composition into the middle ear of a subject.In certain embodiments, administration of the inventive compositionsconsists of applying the composition into the inner ear, sinuses, theeye, vagina, or skin of a subject. In certain embodiments,administration of the inventive compositions consists of applying thecomposition into the sinuses of a subject. In certain embodiments,administration of the inventive compositions consists of applying thecomposition into the eye of a subject. In certain embodiments,administration of the inventive compositions consists of applying thecomposition into the vagina of a subject. In certain embodiments,administration of the inventive compositions consists of applying thecomposition to the skin of a subject. A subject for treatment can be anymammal in need of treatment. In various aspects, the composition is indirect contact with the tympanic membrane for about 1 day to about 30days. In various aspects, the composition is in contact with thetympanic membrane from about 1 day to about 3 days, from about 3 days toabout 7 days, from about 7 days to about 14 days, from about 14 days toabout 21 days, or from about 21 days to about 30 days. In variousembodiments, the composition forms a sustained release reservoir, incontact with the tympanic membrane. In various aspects, the compositionis applied into the ear canal as a liquid, and the composition gels insitu on the surface of the tympanic membrane. When in contact with thetympanic membrane, the therapeutic agent penetrates the tympanicmembrane and is delivered to the middle ear. In various embodiments, thedelivery across the tympanic membrane is a sustained release of thetherapeutic agent over a number of days. The numbers of days that thecomposition can be in contact with the tympanic membrane can be, but isnot limited to, 5 days, 7 days, 10 days, 14 days, 21 days, or 30 days.The composition may be applied singly, or repeatedly in the course oftreatment. In various aspects, the composition may be periodicallyadministered from about every 1 day to about every 7 days, from aboutevery 1 day to about every 14 days, or from about every 1 day to aboutevery 30 days. In various embodiments, the composition is naturallyextruded from the subject at the end of treatment via natural processessimilar to extrusion of ear wax. In certain embodiments, the compositionmay naturally break down, and its degradation products may be eliminatedby the subject. In various embodiments, administration of the inventivecompositions comprises adding the matrix forming agent, the permeationenhancer, and the therapeutic agent to the ear canal; then adding asecond therapeutic agent to the ear canal; and mixing the matrix formingagent, the permeation enhancer, and the therapeutic agent on the earcanal. In certain embodiments, the second therapeutic agent is ananesthetic. In certain embodiments, the second therapeutic agent is alocal anesthetic.

In various embodiments, administration of the inventive compositionscomprises adding the matrix forming agent to the ear canal; adding thepermeation enhancer to the ear canal; adding the therapeutic agent tothe ear canal; and mixing the matrix forming agent, the permeationenhancer, and the therapeutic agent on the ear canal. In variousembodiments, administration of the inventive compositions comprisesadding the matrix forming agent to the ear canal; adding the permeationenhancer to the ear canal; adding the therapeutic agent to the earcanal; adding an additional therapeutic agent to the ear canal; andmixing the matrix forming agent, the permeation enhancer, and thetherapeutic agents on the ear canal. In certain embodiments, adding thetherapeutic agent and adding the permeation enhancer to the ear canalcomprises spraying the therapeutic agent and spraying the permeationenhancer into the ear canal.

In various embodiments, administration of the inventive compositionscomprises adding the therapeutic agent to the ear canal; adding thepermeation enhancer to the ear canal; adding the matrix forming agent tothe ear canal; and mixing the matrix forming agent, the permeationenhancer, and the therapeutic agent on the ear canal. In variousembodiments, administration of the inventive compositions comprisesadding the therapeutic agent to the ear canal; adding an additionaltherapeutic agent to the ear canal; adding the permeation enhancer tothe ear canal; adding the matrix forming agent to the ear canal; andmixing the matrix forming agent, the permeation enhancer, and thetherapeutic agents on the ear canal. In certain embodiments, adding thetherapeutic agent and adding the permeation enhancer to the ear canalcomprises spraying the therapeutic agent and spraying the permeationenhancer into the ear canal. In certain embodiments, the therapeuticagent is an antibiotic or anesthetic agent. In certain embodiments, thetherapeutic agent is an antibiotic. In certain embodiments, thetherapeutic agent is an anesthetic agent. In certain embodiments, thepermeation enhancer is bupivacaine.

In various embodiments, administration of the inventive compositionscomprises adding a composition including one or more therapeutic agents,one or more permeation enhancers, and one or more matrix forming agentsto the ear canal; and subsequently adding a composition comprising notherapeutic agents or one or more therapeutic agents, no permeationenhancers or one or more permeation enhancers, and no matrix formingagents or one or more matrix forming agents to the ear canal. In certainembodiments, the subsequent addition of the one or more therapeuticagents comprises therapeutic agents that are the same as in the firstaddition of the one or more therapeutic agents. In certain embodiments,the subsequent addition of the one or more therapeutic agents comprisestherapeutic agents that are different from those in the first additionof the one or more therapeutic agents. In certain embodiments, thesubsequent addition of permeation enhancers comprises permeationenhancers that are the same as in the first addition of the permeationenhancers. In certain embodiments, the subsequent addition of thepermeation enhancers comprises permeation enhancers that are differentfrom those in the first addition of the permeation enhancers. In certainembodiments, the subsequent addition of matrix forming agents comprisesmatrix forming agents that are the same as in the first addition of thematrix forming agents. In certain embodiments, the subsequent additionof the matrix forming agents comprises matrix forming agents that aredifferent from those in the first addition of the matrix forming agents.In certain embodiments, the time interval between the adding of thefirst composition and second composition is about one minute. In certainembodiments, the time interval between the adding of the firstcomposition and second composition is less than one minute. In certainembodiments, the time interval between the adding of the firstcomposition and second composition is more than one minute.

A dose is determined based on the minimum inhibitory concentrationneeded at the site of infection. Without being bound to a particulartheory, in various aspects the minimum inhibitory concentration for H.influenza or S. pneumoniae middle ear infections is about 4 μg/mL forciprofloxacin. In various aspects, a typical dose will requireapproximately 12 μg of ciprofloxacin, based on an average middle earvolume of 3 mL. In various embodiments, the compositions will comprisesufficient dose to delivery 12 μg of ciprofloxacin to the middle ear. Invarious aspects, the administration of the composition comprises asingle application. In other aspects, the administration of thecomposition comprises multiple applications. For example, thecomposition may be administered two, three, four, or more times. Incertain embodiments, the composition is administered repeatedly untilthe desired clinical outcome is achieved. For example, the infection isresolved. In certain embodiments, the administration of the compositioncomprises a first administration of the composition, followed by asecond administration of the composition after a period of time. Incertain embodiments, the period of time between the first firstadministration of the composition and the second administration of thecomposition is a week. In certain embodiments, the period of timebetween the first first administration of the composition and the secondadministration of the composition is more than one week. In certainembodiments, the period of time between the first first administrationof the composition and the second administration of the composition isone month. In certain embodiments, the period of time between the firstfirst administration of the composition and the second administration ofthe composition is more than one month. In various embodiments,administration of the inventive compositions comprises a firstadministration of a composition without a local anesthetic to the earcanal; followed by a second administration of a composition without alocal anesthetic to the ear canal. In certain embodiments,administration of the inventive compositions comprises a firstadministration of a composition with a local anesthetic to the earcanal; followed by a second administration of a composition without alocal anesthetic to the ear canal.

In various embodiments, administration of the inventive compositionscomprises a first administration of a composition without a localanesthetic to the ear canal; followed by a second administration of acomposition without a permeation enhancer other than a local anestheticto the ear canal. In certain embodiments, administration of theinventive compositions comprises a first administration of a compositionwith a local anesthetic to the ear canal; followed by a secondadministration of a composition without a permeation enhancer other thanlocal anesthetic to the ear canal. In certain embodiments, thecomposition administered first and the composition administered secondare the same. In certain embodiments, the composition administered firstand the composition administered second are different.

Provided herein are methods of delivering a composition of thedisclosure to the surface of tympanic membrane of a subject. In certainembodiments, the subject has an ear disease. In some embodiments, thesubject has otitis media. In some embodiments, the subject is a human.In certain embodiments, the subject is a domesticated animal, such as adog, cat, cow, pig, horse, sheep, or goat.

In certain embodiments, the method of delivering comprises administeringthe composition into the ear canal via an applicator. In certainembodiments, the method of delivering comprises placing drops of thecomposition into the ear canal. In some embodiments, the drops aredelivered from a dropper (e.g., pipet, eye dropper). In someembodiments, the drops are delivered by a syringe. The syringe may beattached to a needle, rigid catheter, or flexible catheter.

In certain embodiments, the method of delivering comprises placing adose of the composition into the ear canal using a catheter. In someembodiments the catheter is attached to a syringe. In some embodiments,the catheter is rigid. In some embodiments the catheter is flexible. Incertain embodiments, the method of delivering comprises placing a doseof the composition into the ear canal using a needle. In someembodiments, the needle is attached to a syringe. In some embodiments,the needle has a blunt tip.

In certain embodiments, the method of delivering comprises placing adose of the composition into the ear canal using a double barrelsyringe. The double barrel syringe may be used to keep two components ofa composition until mixing of the two components occurs duringadministration (e.g., in situ). In some embodiments, the double barrelsyringe is attached to a single catheter or needle. In some embodiments,each barrel of the double barrel syringe is attached to a separateneedle or catheter.

In certain embodiments, the method of treating an infectious disease orear disease comprise instructing a subject to administer, or providinginstructions to a subject for self-administration of, the composition.

In another aspect, provided herein are methods of eradicating a biofilmin a subject comprising administering to a subject in need thereof, acomposition described herein to a subject in need thereof. In anotheraspect, provided herein are methods of eradicating a biofilm comprisingcontacting the biofilm with a composition described herein.

In another aspect, provided herein are methods of inhibiting formationof a biofilm in a subject, comprising administering to a subject in needthereof a composition described herein to a subject in need thereof. Inanother aspect, provided herein are methods of inhibiting formation of abiofilm comprising contacting a surface with a composition describedherein.

Kits

Provided herein are kits comprising any of the compositions describedherein, which may additionally comprise the compositions in sterilepackaging. Provided herein are kits comprising any of the compositionsor matrix-forming agents described herein, which may additionallycomprise the compositions or matrix-forming agents in sterile packaging.The kits may comprise two containers for two-part, matrix-formingagents. The therapeutic agent may be included in one or both of thecontainers of the matrix forming agent, or the therapeutic agent may bepackaged separately. The permeation enhancer may be included in one orboth of the containers of the matrix forming agent, or the permeationenhancer may be packaged separately. In various aspects the kits maycomprise a bottle or bottles, and a dropper or syringe for each bottle.

In certain embodiments, the kit comprises one or more droppers (e.g.,pipet, eye dropper). In certain embodiments, the kit comprises one ormore syringe. In some embodiments, the syringe is pre-loaded with thecomposition, or one or more component of the composition. In certainembodiments, the kit comprises one or more needle (e.g., blunt-tippedneedle). In certain embodiments, the kit comprises one or more catheter(e.g., flexible catheter).

In certain the kit comprises a double barrel syringe. In someembodiments, the double barrel syringe is pre-loaded with two componentsof the composition. In some embodiments, the double barrel syringe isattached to a single catheter or needle. In some embodiments, eachbarrel of the double barrel syringe is attached to a separate needle orcatheter.

In certain embodiments, a kit described herein further includesinstructions for using the kit, such as instructions for using the kitin a method of the disclosure (e.g., instructions for administering acompound or pharmaceutical composition described herein to a subject). Akit described herein may also include information as required by aregulatory agency such as the U.S. Food and Drug Administration (FDA).

Definitions Chemistry Definitions

Definitions of specific functional groups and chemical terms aredescribed in more detail below. The chemical elements are identified inaccordance with the Periodic Table of the Elements, CAS version,Handbook of Chemistry and Physics, 75^(th) Ed., inside cover, andspecific functional groups are generally defined as described therein.Additionally, general principles of organic chemistry, as well asspecific functional moieties and reactivity, are described in OrganicChemistry, Thomas Sorrell, University Science Books, Sausalito, 1999;Smith and March March's Advanced Organic Chemistry, 5^(th) Edition, JohnWiley & Sons, Inc., New York, 2001; Larock, Comprehensive OrganicTransformations, VCH Publishers, Inc., New York, 1989; and Carruthers,Some Modern Methods of Organic Synthesis, 3^(rd) Edition, CambridgeUniversity Press, Cambridge, 1987.

Compounds described herein can comprise one or more asymmetric centers,and thus can exist in various stereoisomeric forms, e.g., enantiomersand/or diastereomers. For example, the compounds described herein can bein the form of an individual enantiomer, diastereomer or geometricisomer, or can be in the form of a mixture of stereoisomers, includingracemic mixtures and mixtures enriched in one or more stereoisomer.Isomers can be isolated from mixtures by methods known to those skilledin the art, including chiral high pressure liquid chromatography (HPLC)and the formation and crystallization of chiral salts; or preferredisomers can be prepared by asymmetric syntheses. See, for example,Jacques et al., Enantiomers, Racemates and Resolutions (WileyInterscience, New York, 1981); Wilen et al., Tetrahedron 33:2725 (1977);Eliel, E. L. Stereochemistry of Carbon Compounds (McGraw-Hill, N Y,1962); and Wilen, S. H. Tables of Resolving Agents and OpticalResolutions p. 268 (E. L. Eliel, Ed., Univ. of Notre Dame Press, NotreDame, Ind. 1972). The invention additionally encompasses compounds asindividual isomers substantially free of other isomers, andalternatively, as mixtures of various isomers.

Unless otherwise stated, structures depicted herein are also meant toinclude compounds that differ only in the presence of one or moreisotopically enriched atoms. For example, compounds having the presentstructures except for the replacement of hydrogen by deuterium ortritium, replacement of ¹⁹F with ¹⁸F, or the replacement of ¹²C with ¹³Cor ¹⁴C are within the scope of the disclosure. Such compounds areuseful, for example, as analytical tools or probes in biological assays.

When a range of values is listed, it is intended to encompass each valueand sub-range within the range. For example “C₁₋₆ alkyl” is intended toencompass, C₁, C₂, C₃, C₄, C₅, C₆, C₁₋₆, C₁₋₅, C₁₋₄, C₁₋₃, C₁₋₂, C₂₋₆,C₂₋₅, C₂₋₄, C₂₋₃, C₃₋₆, C₃₋₅, C₃₋₄, C₄₋₆, C₄₋₅, and C₅₋₆ alkyl.

The term “aliphatic” refers to alkyl, alkenyl, alkynyl, and carbocyclicgroups. Likewise, the term “heteroaliphatic” refers to heteroalkyl,heteroalkenyl, heteroalkynyl, and heterocyclic groups.

The term “alkyl” refers to a radical of a straight-chain or branchedsaturated hydrocarbon group having from 1 to 10 carbon atoms (“C₁₋₁₀alkyl”). In some embodiments, an alkyl group has 1 to 9 carbon atoms(“C₁₋₉ alkyl”). In some embodiments, an alkyl group has 1 to 8 carbonatoms (“C₁₋₈ alkyl”). In some embodiments, an alkyl group has 1 to 7carbon atoms (“C₁₋₇ alkyl”). In some embodiments, an alkyl group has 1to 6 carbon atoms (“C₁₋₆ alkyl”). In some embodiments, an alkyl grouphas 1 to 5 carbon atoms (“C₁₋₅ alkyl”). In some embodiments, an alkylgroup has 1 to 4 carbon atoms (“C₁₋₄ alkyl”). In some embodiments, analkyl group has 1 to 3 carbon atoms (“C₁₋₃ alkyl”). In some embodiments,an alkyl group has 1 to 2 carbon atoms (“C₁₋₂ alkyl”). In someembodiments, an alkyl group has 1 carbon atom (“C₁ alkyl”). In someembodiments, an alkyl group has 2 to 6 carbon atoms (“C₂₋₆ alkyl”).Examples of C₁₋₆ alkyl groups include methyl (C₁), ethyl (C₂), propyl(C₃) (e.g., n-propyl, isopropyl), butyl (C₄) (e.g., n-butyl, tert-butyl,sec-butyl, iso-butyl), pentyl (C₅) (e.g., n-pentyl, 3-pentanyl, amyl,neopentyl, 3-methyl-2-butanyl, tertiary amyl), and hexyl (C₆) (e.g.,n-hexyl). Additional examples of alkyl groups include n-heptyl (C₇),n-octyl (C₈), and the like. Unless otherwise specified, each instance ofan alkyl group is independently unsubstituted (an “unsubstituted alkyl”)or substituted (a “substituted alkyl”) with one or more substituents(e.g., halogen, such as F). In certain embodiments, the alkyl group isan unsubstituted C₁₋₁₀ alkyl (such as unsubstituted C₁₋₆ alkyl, e.g.,—CH₃ (Me), unsubstituted ethyl (Et), unsubstituted propyl (Pr, e.g.,unsubstituted n-propyl (n-Pr), unsubstituted isopropyl (i-Pr)),unsubstituted butyl (Bu, e.g., unsubstituted n-butyl (n-Bu),unsubstituted tert-butyl (tert-Bu or t-Bu), unsubstituted sec-butyl(sec-Bu), unsubstituted isobutyl (i-Bu)). In certain embodiments, thealkyl group is a substituted C₁₋₁₀ alkyl (such as substituted C₁₋₆alkyl, e.g., —CF₃, Bn).

The term “haloalkyl” is a substituted alkyl group, wherein one or moreof the hydrogen atoms are independently replaced by a halogen, e.g.,fluoro, bromo, chloro, or iodo. In some embodiments, the haloalkylmoiety has 1 to 8 carbon atoms (“C₁₋₈ haloalkyl”). In some embodiments,the haloalkyl moiety has 1 to 6 carbon atoms (“C₁₋₆ haloalkyl”). In someembodiments, the haloalkyl moiety has 1 to 4 carbon atoms (“C₁₋₄haloalkyl”). In some embodiments, the haloalkyl moiety has 1 to 3 carbonatoms (“C₁₋₃ haloalkyl”). In some embodiments, the haloalkyl moiety has1 to 2 carbon atoms (“C₁₋₂ haloalkyl”). Examples of haloalkyl groupsinclude —CHF₂, —CH₂F, —CF₃, —CH₂CF₃, —CF₂CF₃, —CF₂CF₂CF₃, —CCl₃, —CFCl₂,—CF₂Cl, and the like.

The term “heteroalkyl” refers to an alkyl group, which further includesat least one heteroatom (e.g., 1, 2, 3, or 4 heteroatoms) selected fromoxygen, nitrogen, or sulfur within (i.e., inserted between adjacentcarbon atoms of) and/or placed at one or more terminal position(s) ofthe parent chain. In certain embodiments, a heteroalkyl group refers toa saturated group having from 1 to 10 carbon atoms and 1 or moreheteroatoms within the parent chain (“heteroC₁₋₁₀ alkyl”). In someembodiments, a heteroalkyl group is a saturated group having 1 to 9carbon atoms and 1 or more heteroatoms within the parent chain(“heteroC₁₋₉ alkyl”). In some embodiments, a heteroalkyl group is asaturated group having 1 to 8 carbon atoms and 1 or more heteroatomswithin the parent chain (“heteroC₁₋₈ alkyl”). In some embodiments, aheteroalkyl group is a saturated group having 1 to 7 carbon atoms and 1or more heteroatoms within the parent chain (“heteroC₁₋₇ alkyl”). Insome embodiments, a heteroalkyl group is a saturated group having 1 to 6carbon atoms and 1 or more heteroatoms within the parent chain(“heteroC_(1_6) alkyl”). In some embodiments, a heteroalkyl group is asaturated group having 1 to 5 carbon atoms and 1 or 2 heteroatoms withinthe parent chain (“heteroC_(1_5) alkyl”). In some embodiments, aheteroalkyl group is a saturated group having 1 to 4 carbon atoms andfor 2 heteroatoms within the parent chain (“heteroC₁₋₄ alkyl”). In someembodiments, a heteroalkyl group is a saturated group having 1 to 3carbon atoms and 1 heteroatom within the parent chain (“heteroC_(1_3)alkyl”). In some embodiments, a heteroalkyl group is a saturated grouphaving 1 to 2 carbon atoms and 1 heteroatom within the parent chain(“heteroC_(1_2) alkyl”). In some embodiments, a heteroalkyl group is asaturated group having 1 carbon atom and 1 heteroatom (“heteroC₁alkyl”). In some embodiments, a heteroalkyl group is a saturated grouphaving 2 to 6 carbon atoms and 1 or 2 heteroatoms within the parentchain (“heteroC₂₋₆ alkyl”). Unless otherwise specified, each instance ofa heteroalkyl group is independently unsubstituted (an “unsubstitutedheteroalkyl”) or substituted (a “substituted heteroalkyl”) with one ormore substituents. In certain embodiments, the heteroalkyl group is anunsubstituted heteroC₁₋₁₀ alkyl. In certain embodiments, the heteroalkylgroup is a substituted heteroC_(1_10) alkyl.

The term “alkenyl” refers to a radical of a straight-chain or branchedhydrocarbon group having from 2 to 10 carbon atoms and one or morecarbon-carbon double bonds (e.g., 1, 2, 3, or 4 double bonds). In someembodiments, an alkenyl group has 2 to 9 carbon atoms (“C₂₋₉ alkenyl”).In some embodiments, an alkenyl group has 2 to 8 carbon atoms (“C₂₋₈alkenyl”). In some embodiments, an alkenyl group has 2 to 7 carbon atoms(“C₂₋₇ alkenyl”). In some embodiments, an alkenyl group has 2 to 6carbon atoms (“C₂₋₆ alkenyl”). In some embodiments, an alkenyl group has2 to 5 carbon atoms (“C₂₋₅ alkenyl”). In some embodiments, an alkenylgroup has 2 to 4 carbon atoms (“C₂₋₄ alkenyl”). In some embodiments, analkenyl group has 2 to 3 carbon atoms (“C₂₋₃ alkenyl”). In someembodiments, an alkenyl group has 2 carbon atoms (“C₂ alkenyl”). The oneor more carbon-carbon double bonds can be internal (such as in2-butenyl) or terminal (such as in 1-butenyl). Examples of C₂₋₄ alkenylgroups include ethenyl (C₂), 1-propenyl (C₃), 2-propenyl (C₃), 1-butenyl(C₄), 2-butenyl (C₄), butadienyl (C₄), and the like. Examples of C₂₋₆alkenyl groups include the aforementioned C₂₋₄ alkenyl groups as well aspentenyl (C₅), pentadienyl (C₅), hexenyl (C₆), and the like. Additionalexamples of alkenyl include heptenyl (C₇), octenyl (C₈), octatrienyl(C₈), and the like. Unless otherwise specified, each instance of analkenyl group is independently unsubstituted (an “unsubstitutedalkenyl”) or substituted (a “substituted alkenyl”) with one or moresubstituents. In certain embodiments, the alkenyl group is anunsubstituted C₂₋₁₀ alkenyl. In certain embodiments, the alkenyl groupis a substituted C₂₋₁₀ alkenyl. In an alkenyl group, a C═C double bondfor which the stereochemistry is not specified (e.g., —CH═CHCH₃ or

may be an (E)- or (Z)-double bond.

The term “heteroalkenyl” refers to an alkenyl group, which furtherincludes at least one heteroatom (e.g., 1, 2, 3, or 4 heteroatoms)selected from oxygen, nitrogen, or sulfur within (i.e., inserted betweenadjacent carbon atoms of) and/or placed at one or more terminalposition(s) of the parent chain. In certain embodiments, a heteroalkenylgroup refers to a group having from 2 to 10 carbon atoms, at least onedouble bond, and 1 or more heteroatoms within the parent chain(“heteroC₂₋₁₀ alkenyl”). In some embodiments, a heteroalkenyl group has2 to 9 carbon atoms at least one double bond, and 1 or more heteroatomswithin the parent chain (“heteroC₂₋₉ alkenyl”). In some embodiments, aheteroalkenyl group has 2 to 8 carbon atoms, at least one double bond,and 1 or more heteroatoms within the parent chain (“heteroC₂₋₈alkenyl”). In some embodiments, a heteroalkenyl group has 2 to 7 carbonatoms, at least one double bond, and 1 or more heteroatoms within theparent chain (“heteroC₂₋₇ alkenyl”). In some embodiments, aheteroalkenyl group has 2 to 6 carbon atoms, at least one double bond,and 1 or more heteroatoms within the parent chain (“heteroC₂₋₆alkenyl”). In some embodiments, a heteroalkenyl group has 2 to 5 carbonatoms, at least one double bond, and 1 or 2 heteroatoms within theparent chain (“heteroC₂₋₅ alkenyl”). In some embodiments, aheteroalkenyl group has 2 to 4 carbon atoms, at least one double bond,and for 2 heteroatoms within the parent chain (“heteroC₂₋₄ alkenyl”). Insome embodiments, a heteroalkenyl group has 2 to 3 carbon atoms, atleast one double bond, and 1 heteroatom within the parent chain(“heteroC₂₋₃ alkenyl”). In some embodiments, a heteroalkenyl group has 2to 6 carbon atoms, at least one double bond, and 1 or 2 heteroatomswithin the parent chain (“heteroC₂₋₆ alkenyl”). Unless otherwisespecified, each instance of a heteroalkenyl group is independentlyunsubstituted (an “unsubstituted heteroalkenyl”) or substituted (a“substituted heteroalkenyl”) with one or more substituents. In certainembodiments, the heteroalkenyl group is an unsubstituted heteroC₂₋₁₀alkenyl. In certain embodiments, the heteroalkenyl group is asubstituted heteroC₂₋₁₀ alkenyl.

The term “alkynyl” refers to a radical of a straight-chain or branchedhydrocarbon group having from 2 to 10 carbon atoms and one or morecarbon-carbon triple bonds (e.g., 1, 2, 3, or 4 triple bonds) (“C₂₋₁₀alkynyl”). In some embodiments, an alkynyl group has 2 to 9 carbon atoms(“C₂₋₉ alkynyl”). In some embodiments, an alkynyl group has 2 to 8carbon atoms (“C₂₋₈ alkynyl”). In some embodiments, an alkynyl group has2 to 7 carbon atoms (“C₂₋₇ alkynyl”). In some embodiments, an alkynylgroup has 2 to 6 carbon atoms (“C₂₋₆ alkynyl”). In some embodiments, analkynyl group has 2 to 5 carbon atoms (“C₂₋₅ alkynyl”). In someembodiments, an alkynyl group has 2 to 4 carbon atoms (“C₂₋₄ alkynyl”).In some embodiments, an alkynyl group has 2 to 3 carbon atoms (“C₂₋₃alkynyl”). In some embodiments, an alkynyl group has 2 carbon atoms (“C₂alkynyl”). The one or more carbon-carbon triple bonds can be internal(such as in 2-butynyl) or terminal (such as in 1-butynyl). Examples ofC₂₋₄ alkynyl groups include, without limitation, ethynyl (C₂),1-propynyl (C₃), 2-propynyl (C₃), 1-butynyl (C₄), 2-butynyl (C₄), andthe like. Examples of C₂₋₆ alkenyl groups include the aforementionedC₂₋₄ alkynyl groups as well as pentynyl (C₅), hexynyl (C₆), and thelike. Additional examples of alkynyl include heptynyl (C₇), octynyl(C₈), and the like. Unless otherwise specified, each instance of analkynyl group is independently unsubstituted (an “unsubstitutedalkynyl”) or substituted (a “substituted alkynyl”) with one or moresubstituents. In certain embodiments, the alkynyl group is anunsubstituted C₂₋₁₀ alkynyl. In certain embodiments, the alkynyl groupis a substituted C₂₋₁₀ alkynyl.

The term “heteroalkynyl” refers to an alkynyl group, which furtherincludes at least one heteroatom (e.g., 1, 2, 3, or 4 heteroatoms)selected from oxygen, nitrogen, or sulfur within (i.e., inserted betweenadjacent carbon atoms of) and/or placed at one or more terminalposition(s) of the parent chain. In certain embodiments, a heteroalkynylgroup refers to a group having from 2 to 10 carbon atoms, at least onetriple bond, and 1 or more heteroatoms within the parent chain(“heteroC₂₋₁₀ alkynyl”). In some embodiments, a heteroalkynyl group has2 to 9 carbon atoms, at least one triple bond, and 1 or more heteroatomswithin the parent chain (“heteroC₂₋₉ alkynyl”). In some embodiments, aheteroalkynyl group has 2 to 8 carbon atoms, at least one triple bond,and 1 or more heteroatoms within the parent chain (“heteroC₂₋₈alkynyl”). In some embodiments, a heteroalkynyl group has 2 to 7 carbonatoms, at least one triple bond, and 1 or more heteroatoms within theparent chain (“heteroC₂₋₇ alkynyl”). In some embodiments, aheteroalkynyl group has 2 to 6 carbon atoms, at least one triple bond,and 1 or more heteroatoms within the parent chain (“heteroC₂₋₆alkynyl”). In some embodiments, a heteroalkynyl group has 2 to 5 carbonatoms, at least one triple bond, and 1 or 2 heteroatoms within theparent chain (“heteroC₂₋₅ alkynyl”). In some embodiments, aheteroalkynyl group has 2 to 4 carbon atoms, at least one triple bond,and for 2 heteroatoms within the parent chain (“heteroC₂₋₄ alkynyl”). Insome embodiments, a heteroalkynyl group has 2 to 3 carbon atoms, atleast one triple bond, and 1 heteroatom within the parent chain(“heteroC₂₋₃ alkynyl”). In some embodiments, a heteroalkynyl group has 2to 6 carbon atoms, at least one triple bond, and 1 or 2 heteroatomswithin the parent chain (“heteroC₂₋₆ alkynyl”). Unless otherwisespecified, each instance of a heteroalkynyl group is independentlyunsubstituted (an “unsubstituted heteroalkynyl”) or substituted (a“substituted heteroalkynyl”) with one or more substituents. In certainembodiments, the heteroalkynyl group is an unsubstituted heteroC₂₋₁₀alkynyl. In certain embodiments, the heteroalkynyl group is asubstituted heteroC₂₋₁₀ alkynyl.

The term “carbocyclyl” or “carbocyclic” refers to a radical of anon-aromatic cyclic hydrocarbon group having from 3 to 14 ring carbonatoms (“C₃₋₁₄ carbocyclyl”) and zero heteroatoms in the non-aromaticring system. In some embodiments, a carbocyclyl group has 3 to 10 ringcarbon atoms (“C₃₋₁₀ carbocyclyl”). In some embodiments, a carbocyclylgroup has 3 to 8 ring carbon atoms (“C₃₋₈ carbocyclyl”). In someembodiments, a carbocyclyl group has 3 to 7 ring carbon atoms (“C₃₋₇carbocyclyl”). In some embodiments, a carbocyclyl group has 3 to 6 ringcarbon atoms (“C₃₋₆ carbocyclyl”). In some embodiments, a carbocyclylgroup has 4 to 6 ring carbon atoms (“C₄₋₆ carbocyclyl”). In someembodiments, a carbocyclyl group has 5 to 6 ring carbon atoms (“C₅₋₆carbocyclyl”). In some embodiments, a carbocyclyl group has 5 to 10 ringcarbon atoms (“C₅₋₁₀ carbocyclyl”). Exemplary C₃₋₆ carbocyclyl groupsinclude, without limitation, cyclopropyl (C₃), cyclopropenyl (C₃),cyclobutyl (C₄), cyclobutenyl (C₄), cyclopentyl (C₅), cyclopentenyl(C₅), cyclohexyl (C₆), cyclohexenyl (C₆), cyclohexadienyl (C₆), and thelike. Exemplary C₃₋₈ carbocyclyl groups include, without limitation, theaforementioned C₃₋₆ carbocyclyl groups as well as cycloheptyl (C₇),cycloheptenyl (C₇), cycloheptadienyl (C₇), cycloheptatrienyl (C₇),cyclooctyl (C₈), cyclooctenyl (C₈), bicyclo[2.2.1]heptanyl (C₇),bicyclo[2.2.2]octanyl (C₈), and the like. Exemplary C₃₋₁₀ carbocyclylgroups include, without limitation, the aforementioned C₃₋₈ carbocyclylgroups as well as cyclononyl (C₉), cyclononenyl (C₉), cyclodecyl (C₁₀),cyclodecenyl (C₁₀), octahydro-1H-indenyl (C₉), decahydronaphthalenyl(C₁₀), spiro[4.5]decanyl (C₁₀), and the like. As the foregoing examplesillustrate, in certain embodiments, the carbocyclyl group is eithermonocyclic (“monocyclic carbocyclyl”) or polycyclic (e.g., containing afused, bridged or spiro ring system such as a bicyclic system (“bicycliccarbocyclyl”) or tricyclic system (“tricyclic carbocyclyl”)) and can besaturated or can contain one or more carbon-carbon double or triplebonds. “Carbocyclyl” also includes ring systems wherein the carbocyclylring, as defined above, is fused with one or more aryl or heteroarylgroups wherein the point of attachment is on the carbocyclyl ring, andin such instances, the number of carbons continue to designate thenumber of carbons in the carbocyclic ring system. Unless otherwisespecified, each instance of a carbocyclyl group is independentlyunsubstituted (an “unsubstituted carbocyclyl”) or substituted (a“substituted carbocyclyl”) with one or more substituents. In certainembodiments, the carbocyclyl group is an unsubstituted C₃₋₁₄carbocyclyl. In certain embodiments, the carbocyclyl group is asubstituted C₃₋₁₄ carbocyclyl.

In some embodiments, “carbocyclyl” is a monocyclic, saturatedcarbocyclyl group having from 3 to 14 ring carbon atoms (“C₃₋₁₄cycloalkyl”). In some embodiments, a cycloalkyl group has 3 to 10 ringcarbon atoms (“C₃₋₁₀ cycloalkyl”). In some embodiments, a cycloalkylgroup has 3 to 8 ring carbon atoms (“C₃₋₈ cycloalkyl”). In someembodiments, a cycloalkyl group has 3 to 6 ring carbon atoms (“C₃₋₆cycloalkyl”). In some embodiments, a cycloalkyl group has 4 to 6 ringcarbon atoms (“C₄₋₆ cycloalkyl”). In some embodiments, a cycloalkylgroup has 5 to 6 ring carbon atoms (“C₅₋₆ cycloalkyl”). In someembodiments, a cycloalkyl group has 5 to 10 ring carbon atoms (“C₅₋₁₀cycloalkyl”). Examples of C₅₋₆ cycloalkyl groups include cyclopentyl(C₅) and cyclohexyl (C₅). Examples of C₃₋₆ cycloalkyl groups include theaforementioned C₅₋₆ cycloalkyl groups as well as cyclopropyl (C₃) andcyclobutyl (C₄). Examples of C₃₋₈ cycloalkyl groups include theaforementioned C₃₋₆ cycloalkyl groups as well as cycloheptyl (C₇) andcyclooctyl (C₈). Unless otherwise specified, each instance of acycloalkyl group is independently unsubstituted (an “unsubstitutedcycloalkyl”) or substituted (a “substituted cycloalkyl”) with one ormore substituents. In certain embodiments, the cycloalkyl group is anunsubstituted C₃₋₁₄ cycloalkyl. In certain embodiments, the cycloalkylgroup is a substituted C₃₋₁₄ cycloalkyl.

The term “heterocyclyl” or “heterocyclic” refers to a radical of a 3- to14-membered non-aromatic ring system having ring carbon atoms and 1 to 4ring heteroatoms, wherein each heteroatom is independently selected fromnitrogen, oxygen, and sulfur (“3-14 membered heterocyclyl”). Inheterocyclyl groups that contain one or more nitrogen atoms, the pointof attachment can be a carbon or nitrogen atom, as valency permits. Aheterocyclyl group can either be monocyclic (“monocyclic heterocyclyl”)or polycyclic (e.g., a fused, bridged or spiro ring system such as abicyclic system (“bicyclic heterocyclyl”) or tricyclic system(“tricyclic heterocyclyl”)), and can be saturated or can contain one ormore carbon-carbon double or triple bonds. Heterocyclyl polycyclic ringsystems can include one or more heteroatoms in one or both rings.“Heterocyclyl” also includes ring systems wherein the heterocyclyl ring,as defined above, is fused with one or more carbocyclyl groups whereinthe point of attachment is either on the carbocyclyl or heterocyclylring, or ring systems wherein the heterocyclyl ring, as defined above,is fused with one or more aryl or heteroaryl groups, wherein the pointof attachment is on the heterocyclyl ring, and in such instances, thenumber of ring members continue to designate the number of ring membersin the heterocyclyl ring system. Unless otherwise specified, eachinstance of heterocyclyl is independently unsubstituted (an“unsubstituted heterocyclyl”) or substituted (a “substitutedheterocyclyl”) with one or more substituents. In certain embodiments,the heterocyclyl group is an unsubstituted 3-14 membered heterocyclyl.In certain embodiments, the heterocyclyl group is a substituted 3-14membered heterocyclyl.

In some embodiments, a heterocyclyl group is a 5-10 memberednon-aromatic ring system having ring carbon atoms and 1-4 ringheteroatoms, wherein each heteroatom is independently selected fromnitrogen, oxygen, and sulfur (“5-10 membered heterocyclyl”). In someembodiments, a heterocyclyl group is a 5-8 membered non-aromatic ringsystem having ring carbon atoms and 1-4 ring heteroatoms, wherein eachheteroatom is independently selected from nitrogen, oxygen, and sulfur(“5-8 membered heterocyclyl”). In some embodiments, a heterocyclyl groupis a 5-6 membered non-aromatic ring system having ring carbon atoms and1-4 ring heteroatoms, wherein each heteroatom is independently selectedfrom nitrogen, oxygen, and sulfur (“5-6 membered heterocyclyl”). In someembodiments, the 5-6 membered heterocyclyl has 1-3 ring heteroatomsselected from nitrogen, oxygen, and sulfur. In some embodiments, the 5-6membered heterocyclyl has 1-2 ring heteroatoms selected from nitrogen,oxygen, and sulfur. In some embodiments, the 5-6 membered heterocyclylhas 1 ring heteroatom selected from nitrogen, oxygen, and sulfur.

Exemplary 3-membered heterocyclyl groups containing 1 heteroatominclude, without limitation, azirdinyl, oxiranyl, and thiiranyl.Exemplary 4-membered heterocyclyl groups containing 1 heteroatominclude, without limitation, azetidinyl, oxetanyl, and thietanyl.Exemplary 5-membered heterocyclyl groups containing 1 heteroatominclude, without limitation, tetrahydrofuranyl, dihydrofuranyl,tetrahydrothiophenyl, dihydrothiophenyl, pyrrolidinyl, dihydropyrrolyl,and pyrrolyl-2,5-dione. Exemplary 5-membered heterocyclyl groupscontaining 2 heteroatoms include, without limitation, dioxolanyl,oxathiolanyl and dithiolanyl. Exemplary 5-membered heterocyclyl groupscontaining 3 heteroatoms include, without limitation, triazolinyl,oxadiazolinyl, and thiadiazolinyl. Exemplary 6-membered heterocyclylgroups containing 1 heteroatom include, without limitation, piperidinyl,tetrahydropyranyl, dihydropyridinyl, and thianyl. Exemplary 6-memberedheterocyclyl groups containing 2 heteroatoms include, withoutlimitation, piperazinyl, morpholinyl, dithianyl, and dioxanyl. Exemplary6-membered heterocyclyl groups containing 3 heteroatoms include, withoutlimitation, triazinyl. Exemplary 7-membered heterocyclyl groupscontaining 1 heteroatom include, without limitation, azepanyl, oxepanyland thiepanyl. Exemplary 8-membered heterocyclyl groups containing 1heteroatom include, without limitation, azocanyl, oxecanyl andthiocanyl. Exemplary bicyclic heterocyclyl groups include, withoutlimitation, indolinyl, isoindolinyl, dihydrobenzofuranyl,dihydrobenzothienyl, tetrahydrobenzothienyl, tetrahydrobenzofuranyl,tetrahydroindolyl, tetrahydroquinolinyl, tetrahydroisoquinolinyl,decahydroquinolinyl, decahydroisoquinolinyl, octahydrochromenyl,octahydroisochromenyl, decahydronaphthyridinyl,decahydro-1,8-naphthyridinyl, octahydropyrrolo[3,2-b]pyrrole, indolinyl,phthalimidyl, naphthalimidyl, chromanyl, chromenyl,1H-benzo[e][1,4]diazepinyl, 1,4,5,7-tetrahydropyrano[3,4-b]pyrrolyl,5,6-dihydro-4H-furo[3,2-b]pyrrolyl, 6,7-dihydro-5H-furo[3,2-b]pyranyl,5,7-dihydro-4H-thieno[2,3-c]pyranyl,2,3-dihydro-1H-pyrrolo[2,3-b]pyridinyl, 2,3-dihydrofuro[2,3-b]pyridinyl,4,5,6,7-tetrahydro-1H-pyrrolo[2,3-b]pyridinyl,4,5,6,7-tetrahydrofuro[3,2-c]pyridinyl,4,5,6,7-tetrahydrothieno[3,2-b]pyridinyl,1,2,3,4-tetrahydro-1,6-naphthyridinyl, and the like.

The term “aryl” refers to a radical of a monocyclic or polycyclic (e.g.,bicyclic or tricyclic) 4n+2 aromatic ring system (e.g., having 6, 10, or14π electrons shared in a cyclic array) having 6-14 ring carbon atomsand zero heteroatoms provided in the aromatic ring system (“C₆₋₁₄aryl”). In some embodiments, an aryl group has 6 ring carbon atoms (“C₆aryl”; e.g., phenyl). In some embodiments, an aryl group has 10 ringcarbon atoms (“C₁₀ aryl”; e.g., naphthyl such as 1-naphthyl and2-naphthyl). In some embodiments, an aryl group has 14 ring carbon atoms(“C₁₄ aryl”; e.g., anthracyl). “Aryl” also includes ring systems whereinthe aryl ring, as defined above, is fused with one or more carbocyclylor heterocyclyl groups wherein the radical or point of attachment is onthe aryl ring, and in such instances, the number of carbon atomscontinue to designate the number of carbon atoms in the aryl ringsystem. Unless otherwise specified, each instance of an aryl group isindependently unsubstituted (an “unsubstituted aryl”) or substituted (a“substituted aryl”) with one or more substituents. In certainembodiments, the aryl group is an unsubstituted C₆₋₁₄ aryl. In certainembodiments, the aryl group is a substituted C₆₋₁₄ aryl.

“Aralkyl” is a subset of “alkyl” and refers to an alkyl groupsubstituted by an aryl group, wherein the point of attachment is on thealkyl moiety.

The term “heteroaryl” refers to a radical of a 5-14 membered monocyclicor polycyclic (e.g., bicyclic, tricyclic) 4n+2 aromatic ring system(e.g., having 6, 10, or 14 r electrons shared in a cyclic array) havingring carbon atoms and 1-4 ring heteroatoms provided in the aromatic ringsystem, wherein each heteroatom is independently selected from nitrogen,oxygen, and sulfur (“5-14 membered heteroaryl”). In heteroaryl groupsthat contain one or more nitrogen atoms, the point of attachment can bea carbon or nitrogen atom, as valency permits. Heteroaryl polycyclicring systems can include one or more heteroatoms in one or both rings.“Heteroaryl” includes ring systems wherein the heteroaryl ring, asdefined above, is fused with one or more carbocyclyl or heterocyclylgroups wherein the point of attachment is on the heteroaryl ring, and insuch instances, the number of ring members continue to designate thenumber of ring members in the heteroaryl ring system. “Heteroaryl” alsoincludes ring systems wherein the heteroaryl ring, as defined above, isfused with one or more aryl groups wherein the point of attachment iseither on the aryl or heteroaryl ring, and in such instances, the numberof ring members designates the number of ring members in the fusedpolycyclic (aryl/heteroaryl) ring system. Polycyclic heteroaryl groupswherein one ring does not contain a heteroatom (e.g., indolyl,quinolinyl, carbazolyl, and the like) the point of attachment can be oneither ring, i.e., either the ring bearing a heteroatom (e.g.,2-indolyl) or the ring that does not contain a heteroatom (e.g.,5-indolyl).

In some embodiments, a heteroaryl group is a 5-10 membered aromatic ringsystem having ring carbon atoms and 1-4 ring heteroatoms provided in thearomatic ring system, wherein each heteroatom is independently selectedfrom nitrogen, oxygen, and sulfur (“5-10 membered heteroaryl”). In someembodiments, a heteroaryl group is a 5-8 membered aromatic ring systemhaving ring carbon atoms and 1-4 ring heteroatoms provided in thearomatic ring system, wherein each heteroatom is independently selectedfrom nitrogen, oxygen, and sulfur (“5-8 membered heteroaryl”). In someembodiments, a heteroaryl group is a 5-6 membered aromatic ring systemhaving ring carbon atoms and 1-4 ring heteroatoms provided in thearomatic ring system, wherein each heteroatom is independently selectedfrom nitrogen, oxygen, and sulfur (“5-6 membered heteroaryl”). In someembodiments, the 5-6 membered heteroaryl has 1-3 ring heteroatomsselected from nitrogen, oxygen, and sulfur. In some embodiments, the 5-6membered heteroaryl has 1-2 ring heteroatoms selected from nitrogen,oxygen, and sulfur. In some embodiments, the 5-6 membered heteroaryl has1 ring heteroatom selected from nitrogen, oxygen, and sulfur. Unlessotherwise specified, each instance of a heteroaryl group isindependently unsubstituted (an “unsubstituted heteroaryl”) orsubstituted (a “substituted heteroaryl”) with one or more substituents.In certain embodiments, the heteroaryl group is an unsubstituted 5-14membered heteroaryl. In certain embodiments, the heteroaryl group is asubstituted 5-14 membered heteroaryl.

Exemplary 5-membered heteroaryl groups containing 1 heteroatom include,without limitation, pyrrolyl, furanyl, and thiophenyl. Exemplary5-membered heteroaryl groups containing 2 heteroatoms include, withoutlimitation, imidazolyl, pyrazolyl, oxazolyl, isoxazolyl, thiazolyl, andisothiazolyl. Exemplary 5-membered heteroaryl groups containing 3heteroatoms include, without limitation, triazolyl, oxadiazolyl, andthiadiazolyl. Exemplary 5-membered heteroaryl groups containing 4heteroatoms include, without limitation, tetrazolyl. Exemplary6-membered heteroaryl groups containing 1 heteroatom include, withoutlimitation, pyridinyl. Exemplary 6-membered heteroaryl groups containing2 heteroatoms include, without limitation, pyridazinyl, pyrimidinyl, andpyrazinyl. Exemplary 6-membered heteroaryl groups containing 3 or 4heteroatoms include, without limitation, triazinyl and tetrazinyl,respectively. Exemplary 7-membered heteroaryl groups containing 1heteroatom include, without limitation, azepinyl, oxepinyl, andthiepinyl. Exemplary 5,6-bicyclic heteroaryl groups include, withoutlimitation, indolyl, isoindolyl, indazolyl, benzotriazolyl,benzothiophenyl, isobenzothiophenyl, benzofuranyl, benzoisofuranyl,benzimidazolyl, benzoxazolyl, benzisoxazolyl, benzoxadiazolyl,benzthiazolyl, benzisothiazolyl, benzthiadiazolyl, indolizinyl, andpurinyl. Exemplary 6,6-bicyclic heteroaryl groups include, withoutlimitation, naphthyridinyl, pteridinyl, quinolinyl, isoquinolinyl,cinnolinyl, quinoxalinyl, phthalazinyl, and quinazolinyl. Exemplarytricyclic heteroaryl groups include, without limitation,phenanthridinyl, dibenzofuranyl, carbazolyl, acridinyl, phenothiazinyl,phenoxazinyl and phenazinyl.

“Heteroaralkyl” is a subset of “alkyl” and refers to an alkyl groupsubstituted by a heteroaryl group, wherein the point of attachment is onthe alkyl moiety.

Affixing the suffix “-ene” to a group indicates the group is a divalentmoiety, e.g., alkylene is the divalent moiety of alkyl, alkenylene isthe divalent moiety of alkenyl, alkynylene is the divalent moiety ofalkynyl, heteroalkylene is the divalent moiety of heteroalkyl,heteroalkenylene is the divalent moiety of heteroalkenyl,heteroalkynylene is the divalent moiety of heteroalkynyl, carbocyclyleneis the divalent moiety of carbocyclyl, heterocyclylene is the divalentmoiety of heterocyclyl, arylene is the divalent moiety of aryl, andheteroarylene is the divalent moiety of heteroaryl.

A group is optionally substituted unless expressly provided otherwise.The term “optionally substituted” refers to being substituted orunsubstituted. In certain embodiments, alkyl, alkenyl, alkynyl,heteroalkyl, heteroalkenyl, heteroalkynyl, carbocyclyl, heterocyclyl,aryl, and heteroaryl groups are optionally substituted. “Optionallysubstituted” refers to a group which may be substituted or unsubstituted(e.g., “substituted” or “unsubstituted” alkyl, “substituted” or“unsubstituted” alkenyl, “substituted” or “unsubstituted” alkynyl,“substituted” or “unsubstituted” heteroalkyl, “substituted” or“unsubstituted” heteroalkenyl, “substituted” or “unsubstituted”heteroalkynyl, “substituted” or “unsubstituted” carbocyclyl,“substituted” or “unsubstituted” heterocyclyl, “substituted” or“unsubstituted” aryl or “substituted” or “unsubstituted” heteroarylgroup). In general, the term “substituted” means that at least onehydrogen present on a group is replaced with a permissible substituent,e.g., a substituent which upon substitution results in a stablecompound, e.g., a compound which does not spontaneously undergotransformation such as by rearrangement, cyclization, elimination, orother reaction. Unless otherwise indicated, a “substituted” group has asubstituent at one or more substitutable positions of the group, andwhen more than one position in any given structure is substituted, thesubstituent is either the same or different at each position. The term“substituted” is contemplated to include substitution with allpermissible substituents of organic compounds, and includes any of thesubstituents described herein that results in the formation of a stablecompound. The present invention contemplates any and all suchcombinations in order to arrive at a stable compound. For purposes ofthis invention, heteroatoms such as nitrogen may have hydrogensubstituents and/or any suitable substituent as described herein whichsatisfy the valencies of the heteroatoms and results in the formation ofa stable moiety. The invention is not intended to be limited in anymanner by the exemplary substituents described herein.

Exemplary carbon atom substituents include, but are not limited to,halogen, —CN, —NO₂, —N₃, —SO₂H, —SO₃H, —OH, —OR^(aa), —ON(R^(bb))₂,—N(R^(bb))₂, —N(R^(bb))₃ ⁺X⁻, —N(OR^(cc))R^(bb), —SH, —SR^(aa),—SSR^(cc), —C(═O)R^(aa), —CO₂H, —CHO, —C(OR^(cc))₂, —CO₂R^(aa),—OC(═O)R^(aa), —OCO₂R^(aa), —C(═O)N(R^(bb))₂, —OC(═O)N(R^(bb))₂,—NR^(bb)C(═O)R^(aa), —NR^(bb)CO₂R^(aa), —NR^(bb)C(═O)N(R^(bb))₂,—C(═NR^(bb))R^(aa), —C(═NR^(bb))OR^(aa), —OC(═NR^(bb))R^(aa),—OC(═NR^(bb))OR^(aa), —C(═NR^(bb))N(R^(bb))₂, —OC(═NR^(bb))N(R^(bb))₂,—NR^(bb)C(═NR^(bb))N(R^(bb))₂, —C(═O)NR^(bb)SO₂R^(aa),—NR^(bb)SO₂R^(aa), —SO₂N(R^(bb))₂, —SO₂R^(aa), —SO₂OR^(aa), —OSO₂R^(aa),—S(═O)R^(aa), —OS(═O)R^(aa), —Si(R^(aa))₃,—OSi(R^(aa))₃—C(═S)N(R^(bb))₂, —C(═O)SR^(aa), —C(═S)SR^(aa),—SC(═S)SR^(aa), —SC(═O)SR^(aa), —OC(═O)SR^(aa), —SC(═O)OR^(aa),—SC(═O)R^(aa), —P(═O)(R^(aa))₂, —P(═O)(OR^(cc))₂, —OP(═O)(R^(aa))₂,—OP(═O)(OR^(cc))₂, —P(═O)(N(R^(bb))₂)₂, —OP(═O)(N(R^(bb))₂)₂,—NR^(bb)P(═O)(R^(aa))₂, —NR^(bb)P(═O)(OR^(cc))₂,—NR^(bb)P(═O)(N(R^(bb))₂)₂, —P(R^(cc))₂, —P(OR^(cc))₂, —P(R^(cc))₃ ⁺X⁻,—P(OR^(cc))₃ ⁺X⁻, —P(R^(cc))₄, —P(OR^(cc))₄, —OP(R^(cc))₂, —OP(R^(cc))₃⁺X⁻, —OP(OR^(cc))₂, —OP(OR^(cc))₃ ⁺X⁻, —OP(R^(cc))₄, —OP(OR^(cc))₄,—B(R^(aa))₂, —B(OR^(cc))₂, —BR^(aa)(OR^(cc)), CMO alkyl, C₁₋₁₀perhaloalkyl, C₂₋₁₀ alkenyl, C₂₋₁₀ alkynyl, heteroC₁₋₁₀ alkyl,heteroC₂₋₁₀ alkenyl, heteroC₂₋₁₀ alkynyl, C₃₋₁₀ carbocyclyl, 3-14membered heterocyclyl, C₆₋₁₄ aryl, and 5-14 membered heteroaryl, whereineach alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl, heteroalkynyl,carbocyclyl, heterocyclyl, aryl, and heteroaryl is independentlysubstituted with 0, 1, 2, 3, 4, or 5 R^(dd) groups; wherein X⁻ is acounterion;

or two geminal hydrogens on a carbon atom are replaced with the group═O, ═S, ═NN(R^(bb))₂, ═NNR^(bb)C(═O)R^(aa), ═NNR^(bb)C(═O)OR^(aa),═NNR^(bb)S(═O)₂R^(aa), ═NR^(bb), or ═NOR^(cc);

each instance of R^(aa) is, independently, selected from C₁₋₁₀ alkyl,C₁₋₁₀ perhaloalkyl, C₂₋₁₀ alkenyl, C₂₋₁₀ alkynyl, heteroC₁₋₁₀ alkyl,heteroC₂₋₁₀alkenyl, heteroC₂₋₁₀alkynyl, C₃₋₁₀ carbocyclyl, 3-14 memberedheterocyclyl, C₆₋₁₄ aryl, and 5-14 membered heteroaryl, or two R^(aa)groups are joined to form a 3-14 membered heterocyclyl or 5-14 memberedheteroaryl ring, wherein each alkyl, alkenyl, alkynyl, heteroalkyl,heteroalkenyl, heteroalkynyl, carbocyclyl, heterocyclyl, aryl, andheteroaryl is independently substituted with 0, 1, 2, 3, 4, or 5 R^(dd)groups;

each instance of R^(bb) is, independently, selected from hydrogen, —OH,—OR^(aa), —N(R^(cc))₂, —CN, —C(═O)R^(aa), —C(═O)N(R^(cc))₂, —CO₂R^(aa),—SO₂R^(aa), —C(═NR^(cc))OR^(aa), —C(═NR^(cc))N(R^(cc))₂, —SO₂N(R^(cc))₂,—SO₂R^(cc), —SO₂OR^(cc), —SO₂R^(aa), —C(═S)N(R^(cc))₂, —C(═O)SR^(cc),—C(═S)SR^(cc), —P(═O)(R^(aa))₂, —P(═O)(OR^(cc))₂, —P(═O)(N(R^(cc))₂)₂,C₁₋₁₀ alkyl, C₁₋₁₀ perhaloalkyl, C₂₋₁₀ alkenyl, C₂₋₁₀ alkynyl,heteroC₁₋₁₀alkyl, heteroC₂₋₁₀alkenyl, heteroC₂₋₁₀alkynyl, C₃₋₁₀carbocyclyl, 3-14 membered heterocyclyl, C₆₋₁₄ aryl, and 5-14 memberedheteroaryl, or two R^(bb) groups are joined to form a 3-14 memberedheterocyclyl or 5-14 membered heteroaryl ring, wherein each alkyl,alkenyl, alkynyl, heteroalkyl, heteroalkenyl, heteroalkynyl,carbocyclyl, heterocyclyl, aryl, and heteroaryl is independentlysubstituted with 0, 1, 2, 3, 4, or 5 R^(dd) groups; wherein X⁻ is acounterion;

each instance of R^(cc) is, independently, selected from hydrogen, C₁₋₁₀alkyl, C₁₋₁₀ perhaloalkyl, C₂₋₁₀ alkenyl, C₂₋₁₀ alkynyl, heteroC₁₋₁₀alkyl, heteroC₂₋₁₀ alkenyl, heteroC₂₋₁₀ alkynyl, C₃₋₁₀ carbocyclyl, 3-14membered heterocyclyl, C₆₋₁₄ aryl, and 5-14 membered heteroaryl, or twoR^(cc) groups are joined to form a 3-14 membered heterocyclyl or 5-14membered heteroaryl ring, wherein each alkyl, alkenyl, alkynyl,heteroalkyl, heteroalkenyl, heteroalkynyl, carbocyclyl, heterocyclyl,aryl, and heteroaryl is independently substituted with 0, 1, 2, 3, 4, or5 R^(dd) groups;

each instance of R^(dd) is, independently, selected from halogen, —CN,—NO₂, —N₃, —SO₂H, —SO₃H, —OH, —OR^(ee), —ON(R^(ff))₂, —N(R^(ff))₂,—N(R^(ff))₃ ⁺X⁻, —N(OR^(ee))R^(ff), —SH, —SR^(ee), —SSR^(ee),—C(═O)R^(ee), —CO₂H, —CO₂R^(ee), —OC(═O)R^(ee), —OCO₂R^(ee),—C(═O)N(R^(ff))₂, —OC(═O)N(R^(ff))₂, —NR^(ff)C(═O)R^(ee),—NR^(ff)CO₂R^(ee), —NR^(ff)C(═O)N(R^(ff))₂, —C(═NR^(ff))OR^(ee),—OC(═NR^(ff))R^(ee), —OC(═NR^(ff))OR^(ee), —C(═NR^(ff))N(R^(ff))₂,—OC(═NR^(ff))N(R^(ff))₂, —NR^(ff)C(═NR^(ff))N(R^(ff))₂,—NR^(ff)SO₂R^(ee), —SO₂N(R^(ff))₂, —SO₂R^(ee), —SO₂OR^(ee), —OSO₂R^(ee),—S(═O)R^(ee), —Si(R^(ee))₃, —OSi(R^(ee))₃, —C(═S)N(R^(ff))₂,—C(═O)SR^(ee), —C(═S)SR^(ee), —SC(═S)SR^(ee), —P(═O)(OR^(ee))₂,—P(═O)(R^(ee))₂, —OP(═O)(R^(ee))₂, —OP(═O)(OR^(ee))₂, C₁₋₆ alkyl, C₁₋₆perhaloalkyl, C₂₋₆ alkenyl, C₂₋₆ alkynyl, heteroC₁₋₆alkyl,heteroC₂₋₆alkenyl, heteroC₂₋₆alkynyl, C₃₋₁₀ carbocyclyl, 3-10 memberedheterocyclyl, C₆₋₁₀ aryl, 5-10 membered heteroaryl, wherein each alkyl,alkenyl, alkynyl, heteroalkyl, heteroalkenyl, heteroalkynyl,carbocyclyl, heterocyclyl, aryl, and heteroaryl is independentlysubstituted with 0, 1, 2, 3, 4, or 5 R^(gg) groups, or two geminalR^(dd) substituents can be joined to form ═O or ═S; wherein X⁻ is acounterion;

-   -   each instance of R^(ee) is, independently, selected from C₁₋₆        alkyl, C₁₋₆ perhaloalkyl, C₂₋₆ alkenyl, C₂₋₆ alkynyl, heteroC₁₋₆        alkyl, heteroC₂₋₆alkenyl, heteroC₂₋₆ alkynyl, C₃₋₁₀ carbocyclyl,        C₆₋₁₀ aryl, 3-10 membered heterocyclyl, and 3-10 membered        heteroaryl, wherein each alkyl, alkenyl, alkynyl, heteroalkyl,        heteroalkenyl, heteroalkynyl, carbocyclyl, heterocyclyl, aryl,        and heteroaryl is independently substituted with 0, 1, 2, 3, 4,        or 5 R^(gg) groups;

each instance of R^(ff) is, independently, selected from hydrogen, C₁₋₆alkyl, C₁₋₆ perhaloalkyl, C₂₋₆ alkenyl, C₂₋₆ alkynyl, heteroC₁₋₆alkyl,heteroC₂₋₆alkenyl, heteroC₂₋₆alkynyl, C₃₋₁₀ carbocyclyl, 3-10 memberedheterocyclyl, C₆₋₁₀ aryl and 5-10 membered heteroaryl, or two R^(ff)groups are joined to form a 3-10 membered heterocyclyl or 5-10 memberedheteroaryl ring, wherein each alkyl, alkenyl, alkynyl, heteroalkyl,heteroalkenyl, heteroalkynyl, carbocyclyl, heterocyclyl, aryl, andheteroaryl is independently substituted with 0, 1, 2, 3, 4, or 5 R^(gg)groups; and

each instance of R^(gg) is, independently, halogen, —CN, —NO₂, —N₃,—SO₂H, —SO₃H, —OH, —OC₁₋₆ alkyl, —ON(C₁₋₆ alkyl)₂, —N(C₁₋₆ alkyl)₂,—N(C₁₋₆ alkyl)₃ ⁺X⁻, —NH(C₁₋₆ alkyl )₂ ⁺X⁻, —NH₂(C₁₋₆ alkyl)⁺X⁻, —NH₃⁺X⁻, —N(OC₁₋₆ alkyl)(C₁₋₆ alkyl), —N(OH)(C₁₋₆ alkyl), —NH(OH), —SH,—SC₁₋₆ alkyl, —SS(C₁₋₆ alkyl), —C(═O)(C₁₋₆ alkyl), —CO₂H, —CO₂(C₁₋₆alkyl), —OC(═O)(C₁₋₆ alkyl), —OCO₂(C₁₋₆ alkyl), —C(═O)NH₂, —C(═O)N(C₁₋₆alkyl)₂, —OC(═O)NH(C₁₋₆ alkyl), —NHC(═O)(C₁₋₆ alkyl), —N(C₁₋₆alkyl)C(═O)(C₁₋₆ alkyl), —NHCO₂(C₁₋₆ alkyl), —NHC(═O)N(C₁₋₆ alkyl)₂,—NHC(═O)NH(C₁₋₆ alkyl), —NHC(═O)NH₂, —C(═NH)O(C₁₋₆ alkyl), —OC(═NH)(C₁₋₆alkyl), —OC(═NH)OC₁₋₆ alkyl, —C(═NH)N(C₁₋₆ alkyl)₂, —C(═NH)NH(C₁₋₆alkyl), —C(═NH)NH₂, —OC(═NH)N(C₁₋₆ alkyl)₂, —OC(NH)NH(C₁₋₆ alkyl),—OC(NH)NH₂, —NHC(NH)N(C₁₋₆ alkyl)₂, —NHC(═NH)NH₂, —NHSO₂(C₁₋₆ alkyl),—SO₂N(C₁₋₆ alkyl)₂, —SO₂NH(C₁₋₆ alkyl), —SO₂NH₂, —SO₂C₁₋₆ alkyl,—SO₂OC₁₋₆ alkyl, —OSO₂C₁₋₆ alkyl, —SOC₁₋₆ alkyl, —Si(C₁₋₆ alkyl)₃,—OSi(C₁₋₆ alkyl)₃-C(═S)N(C₁₋₆ alkyl)₂, C(═S)NH(C₁₋₆ alkyl), C(═S)NH₂,—C(═O)S(C₁₋₆ alkyl), —C(═S)SC₁₋₆ alkyl, —SC(═S)SC₁₋₆ alkyl, —P(═O)(OC₁₋₆alkyl)₂, —P(═O)(C₁₋₆ alkyl)₂, —OP(═O)(C₁₋₆ alkyl)₂, —OP(═O)(OC₁₋₆alkyl)₂, C₁₋₆ alkyl, C₁₋₆ perhaloalkyl, C₂₋₆ alkenyl, C₂₋₆ alkynyl,heteroC₁₋₆alkyl, heteroC₂₋₆alkenyl, heteroC₂₋₆alkynyl, C₃₋₁₀carbocyclyl, C₆₋₁₀ aryl, 3-10 membered heterocyclyl, 5-10 memberedheteroaryl; or two geminal R^(gg) substituents can be joined to form ═Oor ═S; wherein X⁻ is a counterion.

The term “halo” or “halogen” refers to fluorine (fluoro, —F), chlorine(chloro, —Cl), bromine (bromo, —Br), or iodine (iodo, —I).

The term “hydroxyl” or “hydroxy” refers to the group —OH. The term“substituted hydroxyl” or “substituted hydroxyl,” by extension, refersto a hydroxyl group wherein the oxygen atom directly attached to theparent molecule is substituted with a group other than hydrogen, andincludes groups selected from —OR^(aa), —ON(R^(bb))₂, —OC(═O)SR^(aa),—OC(═O)R^(aa), —OCO₂R^(aa), —OC(═O)N(R^(bb))₂, —OC(═NR^(bb))R^(aa),—OC(═NR^(bb))OR^(aa), —OC(═NR^(bb))N(R^(bb))₂, —OS(═O)R^(aa),—OSO₂R^(aa), —OSi(R^(aa))₃, —OP(R^(cc))₂, —OP(R^(cc))₃ ⁺X⁻,—OP(OR^(cc))₂, —OP(OR^(cc))₃ ⁺X⁻, —OP(═O)(R^(aa))₂, —OP(═O)(OR^(cc))₂,and —OP(═O)(N(R^(bb)))₂, wherein X⁻, R^(aa), R^(bb), and R^(cc) are asdefined herein.

The term “amino” refers to the group —NH₂. The term “substituted amino,”by extension, refers to a monosubstituted amino, a disubstituted amino,or a trisubstituted amino. In certain embodiments, the “substitutedamino” is a monosubstituted amino or a disubstituted amino group.

The term “monosubstituted amino” refers to an amino group wherein thenitrogen atom directly attached to the parent molecule is substitutedwith one hydrogen and one group other than hydrogen, and includes groupsselected from —NH(R^(bb)), —NHC(═O)R^(aa), —NHCO₂R^(aa),—NHC(═O)N(R^(bb))₂, —NHC(═NR^(bb))N(R^(bb))₂, —NHSO₂R^(aa),—NHP(═O)(OR^(cc))₂, and —NHP(═O)(N(R^(bb))₂)₂, wherein R^(aa), R^(bb)and R^(cc) are as defined herein, and wherein R^(bb) of the group—NH(R^(bb)) is not hydrogen.

The term “disubstituted amino” refers to an amino group wherein thenitrogen atom directly attached to the parent molecule is substitutedwith two groups other than hydrogen, and includes groups selected from—N(R^(bb))₂, —NR^(bb) C(═O)R^(aa), —NR^(bb)CO₂R^(aa),—NR^(bb)C(═O)N(R^(bb))₂, —NR^(bb)C(═NR^(bb))N(R^(bb))₂,—NR^(bb)SO₂R^(aa), —NR^(bb)P(═O)(OR^(cc))₂, and—NR^(bb)P(═O)(N(R^(bb))₂)₂, wherein R^(aa), R^(bb), and R^(cc) are asdefined herein, with the proviso that the nitrogen atom directlyattached to the parent molecule is not substituted with hydrogen.

The term “trisubstituted amino” refers to an amino group wherein thenitrogen atom directly attached to the parent molecule is substitutedwith three groups, and includes groups selected from —N(R^(bb))₃ and—N(R^(bb))₃ ⁺X⁻, wherein R^(bb) and X⁻ are as defined herein.

The term “acyl” refers to a group having the general formula—C(═O)R^(X1), —C(═O)OR^(X1), —C(═O)—O—C(═O)R^(X1), —C(═O)SR^(X1),—C(═O)N(R^(X1))₂, —C(═S)R^(X1), —C(═S)N(R^(X1))₂, and —C(═S)S(R^(X1)),—C(═NR^(X1))R^(X1), —C(═NR^(X1))OR^(X1), —C(═NR^(X1))SR^(X1), and—C(═NR^(X1))N(R^(X1))₂, wherein R^(X1) is hydrogen; halogen; substitutedor unsubstituted hydroxyl; substituted or unsubstituted thiol;substituted or unsubstituted amino; substituted or unsubstituted acyl,cyclic or acyclic, substituted or unsubstituted, branched or unbranchedaliphatic; cyclic or acyclic, substituted or unsubstituted, branched orunbranched heteroaliphatic; cyclic or acyclic, substituted orunsubstituted, branched or unbranched alkyl; cyclic or acyclic,substituted or unsubstituted, branched or unbranched alkenyl;substituted or unsubstituted alkynyl; substituted or unsubstituted aryl,substituted or unsubstituted heteroaryl, aliphaticoxy,heteroaliphaticoxy, alkyloxy, heteroalkyloxy, aryloxy, heteroaryloxy,aliphaticthioxy, heteroaliphaticthioxy, alkylthioxy, heteroalkylthioxy,arylthioxy, heteroarylthioxy, mono- or di-aliphaticamino, mono- ordi-heteroaliphaticamino, mono- or di-alkylamino, mono- ordi-heteroalkylamino, mono- or di-arylamino, or mono- ordi-heteroarylamino; or two R^(X1) groups taken together form a 5- to6-membered heterocyclic ring. Exemplary acyl groups include aldehydes(—CHO), carboxylic acids (—CO₂H), ketones, acyl halides, esters, amides,imines, carbonates, carbamates, and ureas.

Acyl substituents include, but are not limited to, any of thesubstituents described herein, that result in the formation of a stablemoiety (e.g., aliphatic, alkyl, alkenyl, alkynyl, heteroaliphatic,heterocyclic, aryl, heteroaryl, acyl, oxo, imino, thiooxo, cyano,isocyano, amino, azido, nitro, hydroxyl, thiol, halo, aliphaticamino,heteroaliphaticamino, alkylamino, heteroalkylamino, arylamino,heteroarylamino, alkylaryl, arylalkyl, aliphaticoxy, heteroaliphaticoxy,alkyloxy, heteroalkyloxy, aryloxy, heteroaryloxy, aliphaticthioxy,heteroaliphaticthioxy, alkylthioxy, heteroalkylthioxy, arylthioxy,heteroarylthioxy, acyloxy, and the like, each of which may or may not befurther substituted).

The term “carbonyl” refers a group wherein the carbon directly attachedto the parent molecule is sp² hybridized, and is substituted with anoxygen, nitrogen or sulfur atom, e.g., a group selected from ketones(—C(═O)R^(aa)), carboxylic acids (—CO₂H), aldehydes (—CHO), esters(—CO₂R^(aa), —C(═O)SR^(aa), —C(═S)SR^(aa)), amides (—C(═O)N(R^(bb))₂,—C(═O)NR^(bb)SO₂R^(aa), —C(═S)N(R^(bb))₂), and imines(—C(═NR^(bb))R^(aa), —C(═NR^(bb))OR^(aa)), —C(═NR^(bb))N(R^(bb))₂),wherein R^(aa) and R^(bb) are as defined herein.

The term “oxo” refers to the group ═O, and the term “thiooxo” refers tothe group ═S.

Nitrogen atoms can be substituted or unsubstituted as valency permits,and include primary, secondary, tertiary, and quaternary nitrogen atoms.Exemplary nitrogen atom substituents include, but are not limited to,hydrogen, —OH, —OR^(aa), —N(R^(cc))₂, —CN, —C(═O)R^(aa),—C(═O)N(R^(cc))₂, —CO₂R^(aa), —SO₂R^(aa), —C(═NR^(bb))R^(aa),—C(═NR^(cc))OR^(aa), —C(═NR^(cc))N(R^(cc))₂, —SO₂N(R^(cc))₂, —SO₂R^(cc),—SO₂OR^(cc), —SOR^(aa), —C(═S)N(R^(cc))₂, —C(═O)SR^(cc), —C(═S)SR^(cc),—P(═O)(OR^(cc))₂, —P(═O)(R^(aa))₂, —P(═O)(N(R^(cc))₂)₂, C₁₋₁₀ alkyl, CMOperhaloalkyl, C₂₋₁₀ alkenyl, C₂₋₁₀ alkynyl, heteroC₁₋₁₀alkyl,heteroC₂₋₁₀ alkenyl, heteroC₂₋₁₀alkynyl, C₃₋₁₀ carbocyclyl, 3-14membered heterocyclyl, C₆₋₁₄ aryl, and 5-14 membered heteroaryl, or twoR^(cc) groups attached to an N atom are joined to form a 3-14 memberedheterocyclyl or 5-14 membered heteroaryl ring, wherein each alkyl,alkenyl, alkynyl, heteroalkyl, heteroalkenyl, heteroalkynyl,carbocyclyl, heterocyclyl, aryl, and heteroaryl is independentlysubstituted with 0, 1, 2, 3, 4, or 5 R^(dd) groups, and wherein R^(aa),R^(bb), R^(cc) and R^(dd) are as defined above.

In certain embodiments, the substituent present on an oxygen atom is anoxygen protecting group (also referred to herein as an “hydroxylprotecting group”). Oxygen protecting groups include, but are notlimited to, —R^(aa), —N(R^(bb))₂, —C(═O)SR^(aa), —C(═O)R^(aa),—CO₂R^(aa), —C(═O)N(R^(bb))₂, —C(═NR^(bb))R^(aa), —C(═NR^(bb))OR^(aa),—C(═NR^(bb))N(R^(bb))₂, —S(═O)R^(aa), —SO₂R^(aa), —Si(R^(aa))₃,—P(R^(cc))₂, —P(R^(cc))₃ ⁺X⁻, —P(OR^(cc))₂, —P(OR^(cc))₃ ⁺X⁻,—P(═O)(R^(aa))₂, —P(═O)(OR^(cc))₂, and —P(═O)(N(R^(bb))₂)₂, wherein X⁻,R^(aa), R^(bb), and R^(cc) are as defined herein. Oxygen protectinggroups are well known in the art and include those described in detailin Protecting Groups in Organic Synthesis, T. W. Greene and P. G. M.Wuts, 3^(rd) edition, John Wiley & Sons, 1999, incorporated herein byreference.

In certain embodiments, the substituent present on an oxygen atom is anoxygen protecting group (also referred to herein as an “hydroxylprotecting group”). Oxygen protecting groups include, but are notlimited to, —R^(aa), —N(R^(bb))₂, —C(═O)SR^(aa), —C(═O)R^(aa),—CO₂R^(aa), —C(═O)N(R^(bb))₂, —C(═NR^(bb))R^(aa), —C(═NR^(bb))OR^(aa),—C(═NR^(bb))N(R^(bb))₂, —S(═O)R^(aa), —SO₂R^(aa), —Si(R^(aa))₃,—P(R^(cc))₂, —P(R^(cc))₃ ⁺X⁻, —P(OR^(cc))₂, —P(OR^(cc))₃ ⁺X⁻,—P(═O)(R^(aa))₂, —P(═O)(OR^(cc))₂, and —P(═O)(N(R^(bb))₂)₂, wherein X⁻,R^(aa), R^(bb), and R^(cc) are as defined herein. Oxygen protectinggroups are well known in the art and include those described in detailin Protecting Groups in Organic Synthesis, T. W. Greene and P. G. M.Wuts, 3^(rd) edition, John Wiley & Sons, 1999, incorporated herein byreference.

A “counterion” or “anionic counterion” is a negatively charged groupassociated with a positively charged group in order to maintainelectronic neutrality. An anionic counterion may be monovalent (i.e.,including one formal negative charge). An anionic counterion may also bemultivalent (i.e., including more than one formal negative charge), suchas divalent or trivalent. Exemplary counterions include halide ions(e.g., F⁻, Cl⁻, Br⁻, I⁻), NO₃ ⁻, ClO₄ ⁻, OH⁻, H₂PO₄ ⁻, HCO₃ ⁻, HSO₄ ⁻,sulfonate ions (e.g., methansulfonate, trifluoromethanesulfonate,p-toluenesulfonate, benzenesulfonate, 10-camphor sulfonate,naphthalene-2-sulfonate, naphthalene-1-sulfonic acid-5-sulfonate,ethan-1-sulfonic acid-2-sulfonate, and the like), carboxylate ions(e.g., acetate, propanoate, benzoate, glycerate, lactate, tartrate,glycolate, gluconate, and the like), BF₄ ⁻, PF₄ ⁻, PF₆ ⁻, AsF₆ ⁻, SbF₆⁻, B[3,5-(CF₃)₂C₆H₃)₄)]⁻, B(C₆F₅)₄ ⁻, BPh₄ ⁻, Al(OC(CF₃)₃)₄ ⁻, andcarborane anions (e.g., CB₁₁H₁₂ ⁻ or (HCB₁₁Me₅Br₆)⁻). Exemplarycounterions which may be multivalent include CO₃ ²⁻, HPO₄ ²⁻, PO₄ ³⁻,B₄O₇ ²⁻, SO₄ ²⁻, S₂O₃ ²⁻, carboxylate anions (e.g., tartrate, citrate,fumarate, maleate, malate, malonate, gluconate, succinate, glutarate,adipate, pimelate, suberate, azelate, sebacate, salicylate, phthalates,aspartate, glutamate, and the like), and carboranes.

As used herein, use of the phrase “at least one instance” refers to 1,2, 3, 4, or more instances, but also encompasses a range, e.g., forexample, from 1 to 4, from 1 to 3, from 1 to 2, from 2 to 4, from 2 to3, or from 3 to 4 instances, inclusive.

A “non-hydrogen group” refers to any group that is defined for aparticular variable that is not hydrogen.

The term “polysaccharide” refers to a polymer composed of long chains ofcarbohydrate or monosaccharide units, or derivatives thereof (e.g.,monosaccharides modified to comprise cross-linkable functional groups).Exemplary polysaccharides include, but are not limited to, glycans,glucans, starches, glycogens, arabinoxylans, celluloses, hemicelluloses,chitins, pectins, dextrans, pullulans, chrysolaminarins, curdlans,laminarins, lentinans, lichenins, pleurans, zymosans,glycosaminoglycans, dextrans, hyaluronic acids, chitosans, andchondroitins. The monosaccharide monomers of polysaccharides aretypically connected by glysolidic linkages. Polysaccharides may behydrolyzed to form oligosaccharides, disaccharides, and/or monosaccharides. The term “carbohydrate” or “saccharide” refers to analdehydic or ketonic derivative of polyhydric alcohols. Monosaccharidesare the simplest carbohydrates in that they cannot be hydrolyzed tosmaller carbohydrates. Most monosaccharides can be represented by thegeneral formula C_(y)H_(2y)O_(y) (e.g., C₆H₁₂O₆ (a hexose such asglucose)), wherein y is an integer equal to or greater than 3. Certainpolyhydric alcohols not represented by the general formula describedabove may also be considered monosaccharides. For example, deoxyriboseis of the formula C₅H₁₀O₄ and is a monosaccharide. Monosaccharidesusually consist of five or six carbon atoms and are referred to aspentoses and hexoses, receptively. If the monosaccharide contains analdehyde it is referred to as an aldose; and if it contains a ketone, itis referred to as a ketose. Monosaccharides may also consist of three,four, or seven carbon atoms in an aldose or ketose form and are referredto as trioses, tetroses, and heptoses, respectively. Glyceraldehyde anddihydroxyacetone are considered to be aldotriose and ketotriose sugars,respectively. Examples of aldotetrose sugars include erythrose andthreose; and ketotetrose sugars include erythrulose. Aldopentose sugarsinclude ribose, arabinose, xylose, and lyxose; and ketopentose sugarsinclude ribulose, arabulose, xylulose, and lyxulose. Examples ofaldohexose sugars include glucose (for example, dextrose), mannose,galactose, allose, altrose, talose, gulose, and idose; and ketohexosesugars include fructose, psicose, sorbose, and tagatose. Ketoheptosesugars include sedoheptulose. Each carbon atom of a monosaccharidebearing a hydroxyl group (—OH), with the exception of the first and lastcarbons, is asymmetric, making the carbon atom a stereocenter with twopossible configurations (R or S). Because of this asymmetry, a number ofisomers may exist for any given monosaccharide formula. The aldohexoseD-glucose, for example, has the formula C₆H₁₂O₆, of which all but two ofits six carbons atoms are stereogenic, making D-glucose one of the 16(i.e., 2⁴) possible stereoisomers. The assignment of D or L is madeaccording to the orientation of the asymmetric carbon furthest from thecarbonyl group: in a standard Fischer projection if the hydroxyl groupis on the right the molecule is a D sugar, otherwise it is an L sugar.The aldehyde or ketone group of a straight-chain monosaccharide willreact reversibly with a hydroxyl group on a different carbon atom toform a hemiacetal or hemiketal, forming a heterocyclic ring with anoxygen bridge between two carbon atoms. Rings with five and six atomsare called furanose and pyranose forms, respectively, and exist inequilibrium with the straight-chain form. During the conversion from thestraight-chain form to the cyclic form, the carbon atom containing thecarbonyl oxygen, called the anomeric carbon, becomes a stereogeniccenter with two possible configurations: the oxygen atom may take aposition either above or below the plane of the ring. The resultingpossible pair of stereoisomers is called anomers. In an a anomer, the—OH substituent on the anomeric carbon rests on the opposite side(trans) of the ring from the —CH₂OH side branch. The alternative form,in which the —CH₂OH substituent and the anomeric hydroxyl are on thesame side (cis) of the plane of the ring, is called a β anomer. The termcarbohydrate also includes other natural or synthetic stereoisomers ofthe carbohydrates described herein.

These and other exemplary substituents are described in more detail inthe Detailed Description, Examples, and Claims. The invention is notintended to be limited in any manner by the above exemplary listing ofsubstituents.

Other Definitions

Animal: The term animal, as used herein, refers to humans as well asnon-human animals, including, for example, mammals, birds, reptiles,amphibians, and fish. Preferably, the non-human animal is a mammal(e.g., a rodent, a mouse, a rat, a rabbit, a monkey, a dog, a cat, aprimate, or a pig). A non-human animal may be a transgenic animal.

Approximately or About: As used herein, the terms “approximately” or“about” in reference to a number are generally taken to include numbersthat fall within a range of 5%, 10%, 15%, or 20% in either direction(greater than or less than) of the number unless otherwise stated orotherwise evident from the context (except where such number would beless than 0% or exceed 100% of a possible value).

Biocompatible: As used herein, the term “biocompatible” refers tosubstances that are not toxic to cells. In some embodiments, a substanceis considered to be “biocompatible” if its addition to cells in vivodoes not induce inflammation and/or other adverse effects in vivo. Insome embodiments, a substance is considered to be “biocompatible” if itsaddition to cells in vitro or in vivo results in less than or equal toabout 50%, about 45%, about 40%, about 35%, about 30%, about 25%, about20%, about 15%, about 10%, about 5%, or less than about 5% cell death.

Biodegradable: As used herein, the term “biodegradable” refers tosubstances that are degraded under physiological conditions. In someembodiments, a biodegradable substance is a substance that is brokendown by cellular machinery. In some embodiments, a biodegradablesubstance is a substance that is broken down by chemical processes.

Optically transparent: As used herein, the term “optically transparent”refers to substances through which light passes through with little orno light being absorbed or reflected. In some embodiments, opticallytransparent refers to substances through which light passes through withno light being absorbed or reflected. In some embodiments, opticallytransparent refers to substances through which light passes through withlittle light being absorbed or reflected. In some embodiments, anoptically transparent substance is substantially clear. In someembodiments, an optically transparent substance is clear.

Effective amount: In general, the “effective amount” of an active agentrefers to an amount sufficient to elicit the desired biologicalresponse. As will be appreciated by those of ordinary skill in this art,the effective amount of a compound of the invention may vary dependingon such factors as the desired biological endpoint, the pharmacokineticsof the compound, the disease being treated, the mode of administration,and the patient. The effective amount of a compound used to treatinfection is the amount needed to kill or prevent the growth of theorganism(s) responsible for the infection.

In vitro: As used herein, the term “in vitro” refers to events thatoccur in an artificial environment, e.g., in a test tube or reactionvessel, in cell culture, etc., rather than within an organism (e.g.animal, plant, and/or microbe).

In vivo: As used herein, the term “in vivo” refers to events that occurwithin an organism (e.g. animal, plant, and/or microbe).

Suffering from: An individual who is “suffering from” a disease,disorder, and/or condition has been diagnosed with or displays one ormore symptoms of the disease, disorder, and/or condition.

Treating: As used herein, the term “treating” refers to partially orcompletely alleviating, ameliorating, relieving, delaying onset of,inhibiting progression of, reducing severity of, and/or reducingincidence of one or more symptoms or features of a particular disease,disorder, and/or condition. For example, “treating” a microbialinfection may refer to inhibiting survival, growth, and/or spread of themicrobe. Treatment may be administered to a subject who does not exhibitsigns of a disease, disorder, and/or condition and/or to a subject whoexhibits only early signs of a disease, disorder, and/or condition forthe purpose of decreasing the risk of developing pathology associatedwith the disease, disorder, and/or condition. In some embodiments,treatment comprises delivery of an inventive vaccine nanocarrier to asubject.

Therapeutic agent: Also referred to as a “drug” is used herein to referto an agent that is administered to a subject to treat a disease,disorder, or other clinically recognized condition that is harmful tothe subject, or for prophylactic purposes, and has a clinicallysignificant effect on the body to treat or prevent the disease,disorder, or condition. Therapeutic agents include, without limitation,agents listed in the United States Pharmacopeia (USP), Goodman andGilman's The Pharmacological Basis of Therapeutics, 10^(th) Ed., McGrawHill, 2001; Katzung, B. (ed.) Basic and Clinical Pharmacology,McGraw-Hill/Appleton & Lange; 8th edition (Sep. 21, 2000); Physician'sDesk Reference (Thomson Publishing), and/or The Merck Manual ofDiagnosis and Therapy, 17^(th) ed. (1999), or the 18th Ed. (2006)following its publication, Mark H. Beers and Robert Berkow (Eds.), MerckPublishing Group, or, in the case of animals, The Merck VeterinaryManual, 9^(th) ed., Kahn, C. A. (Ed.), Merck Publishing Group, 2005.

Diagnostic agent: As used herein, the term “diagnostic agent” refers toan agent that is administered to a subject to aid in the diagnosis of adisease, disorder, or condition. In some embodiments, a diagnostic agentis used to define and/or characterize the localization of a pathologicalprocess. Diagnostic agents include X-ray contrast agents, radioactiveisotopes, and dyes.

Surfactant: As used herein, the term “surfactant” refers to any agentwhich preferentially absorbs to an interface between two immisciblephases, such as the interface between water and an organic solvent, awater/air interface, or an organic solvent/air interface. Surfactantsusually possess a hydrophilic moiety and a hydrophobic moiety.Surfactants may also promote flux of a therapeutic or diagnostic agentacross a biological membrane, e.g., a tympanic membrane.

Terpenes: As used herein, the term “terpene” refers to any agentderived, e.g., biosynthetically, or thought to be derived from unit(s)of isoprene (a five carbon unit). For example, isoprene units ofterpenes may be linked together to form linear chains or they may bearranged to form rings. Typically, the terpenes disclosed herein promoteflux of a therapeutic or diagnostic agent across a biological membrane,e.g., a tympanic membrane. Terpenes may be naturally derived orsynthetically prepared.

The terms “composition” and “formulation” are used interchangeably.

EXAMPLES

In order that the invention described herein may be more fullyunderstood, the following examples are set forth. The examples describedin this application are offered to illustrate the compounds,pharmaceutical compositions, and methods provided herein and are not tobe construed in any way as limiting their scope.

Materials and Methods

Method and Design: The experiments compared the effect of the polymermatrix and incorporation of CPEs on TM permeability and OM cure rate.For the ex vivo experiments, a sample size of 4 for each formulation waschosen, which would provide 80% power to detect 50% differences in fluxbased on power analysis using the nonparametric Friedman test (version7.0, nQuery Advisor, Statistical Solutions, Saugus, Mass.). Sample sizesof 8-10 were used for the in vivo experiments, which were supported byprevious publications (Pelton et al., Antimicrob. Agents Chemother. 44,654-657 (2000)). Comparisons between positive and negative efficacyresults were assessed using Fisher's exact test. Statistical analysiswas conducted using SAS software (version 9.2, SAS Institute, Cary,N.C.). Two-tailed p<0.05 with appropriate Bonferroni-Sidak adjustmentfor multiple comparisons were considered statistically significant inorder to control type I error. During ex vivo experiments, datacollection was stopped after 48 hours due to microbial growth onharvested TM; whereas during in vivo experiments, data collection wasstopped after 7 days because OM would either be cleared or cause theanimal severe illness that requires euthanasia. In vivo experiments wereblinded. All experiments were randomized.

Materials: 2-chloro-2-oxo-1,3,2-dioxaphospholane (COP),1,8-diazabicyclo[5.4.0]undec-7-ene (DBU), n-butanol, diethyl ether,acetic acid, anhydrous dichloromethane, anhydrous tetrahydrofuran wereused as received from Sigma-Aldrich Company (St. Louis, Mo.). Kolliphor®P 407 microprilled (poloxamer 407), received from BASF (Florham Park,N.J.).

Animal maintenance: Healthy adult male chinchillas weighting 500 to 650g were purchased from Ryerson Chinchilla Ranch (Plymouth, Ohio) and carefor in accordance with protocols approved institutionally andnationally. Experiments were carried out in accordance with the BostonChildren's Hospital, Boston University Medical Center, and MassachusettsEye and Ear Infirmary Animal Use Guidelines and approved by eachinstitution's Animal Care and Use Committee.

Hydrogel formation: P407-PBP hydrogel formulations were made by addingpowdered polymers to aqueous solutions. Gels of varying P407-PBP weightpercentage (10% to 18%) can be prepared by simple dissolution in a coldroom to allow better solubility.

Gelation time: Hydrogel formulation in scintillation vials were immersedin a water bath kept at 37° C. with continuous stirring (200 rpm). Thetime it took the stir bar to stop rotating after immersion was recordedas the gelation time.

Gelation temperature: Gelation temperature was quantified using linearoscillatory shear rheology measurements (100 rads⁻¹, 1% strain, 1° C.min⁻¹). Gelation temperature was taken as the temperature at which thestorage modulus (G′) becomes greater than the loss modulus (G″). Thechanges of G′ and G″ over temperatures ranging from 0° C. to above bodytemperature were recorded to reflect changes in mechanical properties.

In vitro release studies: The release of ciprofloxacin from eachformulation was measured using a diffusion system. Transwell® membraneinserts (0.4 μm pore size, 1.1 cm² area; Costar, Cambridge, Mass.) and24-well culture plates were employed as the donor and acceptor chambers,respectively. 200 μL of each formulation was pipetted directly ontopre-warmed filter inserts to obtain a solid hydrogel. Filter inserts(donor compartments) with formed gels were suspended in wells (acceptorcompartments) filled with pre-warmed phosphate buffered saline (PBS) andthe plates then incubated in a 37° C. oven. At each time point (0.5, 1,2, 6, 12, 24, 48 h), 1 mL aliquots of the PBS receiving media weresampled and inserts sequentially moved into a new well with fresh PBS.Aliquots were suspended in 70:30 acetonitrile/PBS to ensure total drugdissolution. Sample aliquots were chromatographically analyzed with HPLCto determine ciprofloxacin concentrations (X, =275 nm). More detailedregarding the ciprofloxacin measurement and HPLC conditions can be foundin reference (8). Experiments were performed in quadruplicate.

Ex vivo permeation experiment: The cross-TM permeation rate ofciprofloxacin was determined with auditory bullae harvested from healthychinchillas. All formulations were applied into the bullae kept at 37°C. and deposited onto the TMs. The volume applied was 200 μL, whichtranslates to 2 mg ciprofloxacin. Permeation of ciprofloxacin across TMinto the receiving chamber was quantified using HPLC. Detailedinformation regarding TM harvesting, TM electrical resistancemeasurement, and configuration of the ex vivo permeation experiment canbe found in reference (8).

Cytotoxicity analysis: Cell viabilities were evaluated with an assay ofa mitochondrial metabolic activity, the CellTiter 96® Aqueous OneSolution Cell Proliferation Assay (Promega Corp.) that uses atetrazolium compound[3-(4,5-dimethyl-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium,inner salt; MTS] and an electron coupling reagent (phenazineethosulfate; PES). On days 1 and 3 of the culture, human dermalfibroblasts (hFB), PC12, and normal adult human primary epidermalkeratinocytes (ATCC) were incubated with CellTiter 96® Aqueous OneSolution for 120 min at 37° C. The absorbance of the culture medium at490 nm was immediately recorded with a 96-well plate reader. Thequantity of formazan product (converted from tetrazole) as measured bythe absorbance at 490 nm is directly proportional to cell metabolicactivity in culture. Planar cultures on 24-well plate were used ascontrols. For each group, n=4. Cell viability was confirmed using aLIVE/DEAD® Viability/Cytotoxicity Kit (Molecular Probes, Invitrogen).Cells were incubated with 1 μM calcein-AM and 2 μM ethidium homodimer-1(EthD-1) for 30 min at 37° C. to label live and dead cells,respectively. Cell viability was calculated as live/(live+dead)×100.

Histopathology: Formulations were administered to the ear canals of livehealthy/OM chinchillas. Seven days later, they were euthanized asdescribed elsewhere (8). Following sacrifice, the TMs were excised andimmediately fixed in 10% neutral buffered formalin overnight, thendecalcified, embedded in paraffin, sectioned (5 μm thick) and stainedwith hematoxylin and eosin by the Department of Pathology at BostonChildren's Hospital (fee for service), using standard techniques. Allstained specimens were evaluated under light microscopy (OlympusFSX-100).

Auditory brainstem response (ABR) measurements: ABR experiments wereconducted with a custom-designed stimulus generation and measurementsystem built around National Instruments (Austin, Tex.) software (LabView) and hardware. Detailed information regarding ABR can be found inreference (8).

NTHi OM Model and pharmacokinetics: All procedures and manipulationswere performed using sedation analgesia with a mixture of ketamine andxylazine given intramuscularly in accordance with approved IACUCprotocols at Boston University Medical Center. Baseline plasma sampleswere obtained through the cephalic sinus 24 hours prior to bacterialinoculation. Isolates of NTHi grown to the mid-log phase were diluted inHBSS, and approximately 25-75 cfu in 100 μL was introduced directly intoeach middle ear bulla under aseptic conditions. Daily tympanometry andotomicroscopy were performed to determine the presence of fluid in theauditory bullae and signs of infection including bulging tympanicmembrane. Erythema and pictures were taken. Once abnormality wasidentified, the middle ear cavity was accessed 48 to 72 h later asdescribed previously (See Sabharwal et al., Infect. Immun. 77, 1121-1127(2009)). A direct culture of middle ear was obtained with a calciumalginate swab and immediately streaked onto a blood agar plate. Middleear fluid was obtained with a 22-gauge angiocatheter connected to anempty tuberculin syringe, 10-20 μL of middle ear fluid was diluted 1:10in HBSS, and three serial 10-fold dilutions were prepared. One hundredmicroliters of each dilution was plated onto blood agar. The lower limitof detection of viable organisms in middle ear fluid using this dilutionseries was 100 cfu mL⁻¹. Direct and indirect ear examination wasperformed every 1 to 2 days until the middle-ear cultures were sterile.Serial plasma samples were obtained during the experiment to determinesystemic drug levels.

Statistical Analysis: Data which were Normally Distributed wereDescribed with Means and Standard Deviations and Compared by UnpairedStudent t-Tests. Otherwise, Data were Presented as Median±Quartiles. AllData Analyses were Performed Using Origin 8 Software. Methods andResults

Isolation of Intact Chinchilla TMs.

The size of the tympanic membrane, middle ear structures, and auditoryrange of chinchillas closely approximate those of humans. A reproducibleex vivo method for studying flux across the tympanic membrane (TM) hasbeen established. TMs were removed undamaged, with the bony tympanicring still attached. Their integrity was assessed by measuring theirelectrical resistance (indicated by RA≥18 kOhm*cm²) in a setup where TMswere placed horizontally in a 12-well plate with donor solution aboveand recipient solution below. The same set-up was used to measure drugflux, in lieu of a conventional diffusion cell—which would deform orrupture the TM. Skin samples, which had worse reproducibility than TMs,were only used as screening tools, to minimize the use of animals.

Trans-Tympanic Delivery of Antibiotics.

For trans-tympanic delivery of antibiotics ciprofloxacin, a syntheticfluoroquinolone antibiotic, was selected because of its known activityagainst non-typable Haemophilus influenzae (NTHi) and Streptococcuspneumoniae (SP), its low molecular weight and moderate lipophilicity.

CPEs Enhance Drug Flux Across the Intact TM.

Sodium dodecyl sulfate (SDS; anionic surfactant), and limonene(monocyclic terpene) were selected as chemical permeation enhancers(CPEs) based on their use in transdermal drug delivery and theirfavorable enhancement/irritation ratio. [28] Bupivacaine, an amino amidelocal anesthetic, was incorporated in some formulations for itspotential clinical benefit to OM-associated otalgia, and becauseamino-ester anesthetics (e.g. tetracaine) act as CPEs. [15] In theabsence of CPEs, ciprofloxacin permeation across the chinchilla TM at37° C. was undetectable up to 12 hours. At 24 hours, 109 μg (out of 2 mgtotal ciprofloxacin), or 5.5% of the starting drug load, had permeatedthe TM; at 48 hours 364 μg (18%) had done so. The addition of limoneneaccelerated drug permeation; ciprofloxacin was detected in the receivingbuffer in as little as 1-2 hours. A two-to-three-foldconcentration-dependent increase in ciprofloxacin transfer at 48 hourswas also achieved. Ciprofloxacin permeation was further enhanced by theuse of all three CPEs together (1% SDS, 0.5% bupivacaine, and 2%limonene; termed 3CPE).

Hydrogels at the TM.

The hydrogel component, Poloxamer 407 (P407) served to hold the drug-CPEcombination in place at the TM for the duration of experiment.Drug-loaded 18% P407 formulations formed soft, clear gels when depositedon chinchilla TMs at 37° C. The hydrogel matrix slowed thetrans-tympanic transfer of ciprofloxacin. (FIG. 3). The addition of 3CPEincreased flux (but still not to the level of ciprofloxacin+CPE withoutgel), so that 3 μg of ciprofloxacin crossed the TM after 6 hours and 14μg crossed after 12 hours (FIG. 3) This increase was seen at all timepoints, with 3CPE almost doubling the amount of ciprofloxacin crossingthe TM in 120 hours (812 μg vs. 441 μg). The formulation ofciprofloxacin in 18% P407 with 3CPE is termed the standard formulationbelow.

Biocompatibility.

In vivo, TMs exposed to ciprofloxacin-loaded gels without 3CPE for 7days were mildly edematous but without inflammation (FIG. 2). Slightlymore pronounced edema was seen in tissue exposed to ciprofloxacin-loadedgels with 3 CPEs, but again tissue reaction was benign. In contrast, TMsextracted after 7 days of untreated H. Influenzae infection wereapproximately five times thicker and exhibited a prominent neutrophilicinflammatory response.

Measurement of Acoustic Brainstem Response (ABR).

Drug-CPE-hydrogels should not affect hearing thresholds or be ototoxic.ABR thresholds after application of the gel-enhancer formulation weresimilar to pre-application measurements (FIG. 4).

The Chinchilla Model of OM.

The infectious inoculum is placed in the middle ear through the superiorbullae, so that there is no open portal for infection through a TMinjury, and drug flux across the TM will be unaffected by theinoculation itself. 100% of animals treated in this manner with S.pneumoniae (SP) and non-typable H. influenzae (NTHi) develop OM. Instudies with a single strain of NTHi, OM resolved in approximately 50%of animals treated with the standard formulation (vs. 20% for untreatedanimals). Ciprofloxacin was undetectable in the blood.

The relatively low cure rate likely reflected inadequate drug flux invivo, and may be attributable to the following factors. 1) Inadequatedrug loading and/or CPE loading. 2) Poor mechanical properties of thegel. At 27° C., the incorporation of CPEs changed the phase transitionof P407 solution (FIG. 5) so that the storage modulus did not becomegreater than the loss modulus, i.e., gelation did not occur. Whilegelation still occurred at 37° C., these data show that the gelation wasnot mechanically robust. This view is consistent with a finding onotoscopy that the P407-based gels were spread out in the auditory canal;lack of bioadhesiveness is another possible contributing factor. Aseparate issue is that gelation took ˜20 sec. This may be adequate inanesthetized animals, but not in active toddlers.

Synthesis of a P407-PPE Polymer.

The hydrophobic monomer, 2-butoxy-2-oxo-1,3,2-dioxaphospholane (BP) wasprepared by condensation reaction of2-chloro-2-oxo-1,3,2-dioxaphospholane (COP) and butanol, then purifiedby vacuum distillation, and analyzed by proton and phosphorous NMRspectroscopy. Hydrophobic P407-PPE polymer (PBP-P407-PBP) wassynthesized by ring opening polymerization (ROP) of BP with P407 in thepresence of an organocatalyst, 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU)at −20° C. Upon completion of the reaction (complete monomer consumptionconfirmed by NMR spectroscopy), excess acetic acid in dichloromethane(DCM) was added to the reaction mixture to quench the reaction. Theproduct was purified by precipitation into ether (3 times) and dried toa white powder under vacuum. Proton and phosphorous NMR spectroscopy,Fourier transform infrared spectroscopy (FTIR), and gel permeationchromatography were used to characterize the polymer and confirm itspurity. FTIR spectroscopy of the product, PBP-P407-PBP, and startingmaterial, P407, are compared in FIG. 9. The molecular weight of thecopolymer was measured as 30.2 kDa by gel permeation chromatography.

Synthesis of P407-PPE Polymers.

The pentablock copolymer P407-polybutylphosphoester with n-butyl (FIG.11A) or s-butyl side groups (FIG. 11B) was synthesized via ring-openingpolymerization (ROP) in the presence of an organocatalyst,1,8-diazabicyclo[5.4.0]undec-7-ene (DBU), at −20° C. After purificationby precipitation, Fourier transform infrared spectroscopy (FTIR, FIG.11A-11B) showed peaks near 1650 and 1050 cm⁻¹ which are characteristicof the stretching of ═P—O and P—O respectively in thepolybutylphosphoester (PBP) moieties (Lin-Vien et al., The Handbook ofInfrared and Raman Characteristic Frequencies of Organic Molecules(Academic Press, New York) (1991)). Both were present in the spectrum ofthe ROP product but not the reactant P407, indicating the successfuladdition of PPE blocks. Increased molecular weight, demonstrated by gelpermeation chromatography (GPC, table 51), indicated that P407 waschemically modified by n-butyl PBP or s-butyl PBP blocks, and not aphysical mixture. The number averaged molecular weight (M_(n)) measuredby nuclear magnetic resonance (NMR) agreed with that calculated from thechemical formulae (table 51). M_(n) measured by GPC was ˜30% higher thanthat measured using NMR; that discrepancy is well documented in theliterature (Wong et al., ACS Macro Lett. 1, 1266-1269 (2012)). In thesynthetic scheme, a small degree of polymerization (DP) was targeted toavoid gelation at or below room temperature and to facilitatedegradation of the hydrogel. The actual DP of the PPE blocks wasdetermined to be 5 using NMR (FIG. 12A). FIG. 12A depicts the nuclearmagnetic resonance (NMR) of pentablock copolymers. DP was calculatedusing the ratio of peak areas at 0.90 ppm (monomer side chain) and 1.14ppm (P407 backbone). The chemical shifts (6, in ppm) for the peakscorresponding to the hydrogens in italics in the following list ofpolymers is provided below. t/m/broad indicate the shape of a peak(i.e., triplet, multiple, broad). CDCl₃ was the solvent. For P407-PBPwith n-butyl groups, ¹H NMR (CDCl₃, ppm): δ 0.90-0.96 (t, 3H,CH₂CH₂CH₂CH₃), 1.14 (m, 3H, CH₂CH(CH₃)O), 1.36-1.46 (m, 2H,CH₂CH₂CH₂CH₃), 1.62-1.72 (m, 2H, CH₂CH₂CH₂CH₃), 3.36-3.42 (m,CH₂CH(CH₃)O), 3.48-3.58 (m, 2H, CH₂CH(CH₃)O), 3.65 (m, 4H, OCH₂CH₂O),4.04-4.14 (m, 2H, PCH₂CH₂CH₂CH₃), 4.16-4.30 (broad, 4H, POCH₂CH₂O). ForP407-PBP with s-butyl groups, ¹H NMR (CDCl₃, ppm): δ 0.90-0.96 (t, 3H,CH₃CHCH₂CH₃), 1.13 (m, 3H, CH₂CH(CH₃)O), 1.30-1.34 (b, 3H, CH₃CHCH₂CH₃),1.55-1.73 (m, 2H, CH₃CHCH₂CH₃), 3.35-3.42 (m, CH₂CH(CH₃)O), 3.48-3.58(m, 2H, CH₂CH(CH₃)O), 3.65 (m, 4H, OCH₂CH₂O), 4.11-4.19 (m, 1H,CH₃CHCH₂CH₃), 4.19-4.35 (broad, 4H, POCH₂CH₂O). Smaller DP, such asDP=2.5, resulted in a high gelation temperature and poor shear strength(FIG. 12B). P407-PBP with an n-butyl group and a DP of 5 was used insubsequent studies because it gelled at a lower temperature and withgreater shear moduli than those made with s-butyl groups (FIG. 12B).

TABLE S1 Molecular weight measured by GPC and NMR and polydispersityindices of P407, and pentablock copolymers P407- PBP with n- or s-butylgroups. P407-PBP P407-PBP P407-PBP n-butyl s-butyl s-butyl P407 DP = 5DP = 5 DP = 2.5 Weight average molecular 18.0 23.7 23.5 21.2 weight,M_(w) (kDa) Number average molecular 15.1 19.1 18.6 17.3 weight, M_(n)(kDa) M_(n) calculated using NMR n.a. 13.9 13.9 13.4 (kDa)Polydispersity index, M_(w)/M_(n)  1.2 1.2 1.3 1.2

Gel Properties of P407-PPE Polymer

A 18% aqueous solution of commercially available P407 (i.e., 18%[P407]), which demonstrated reverse thermal gelation, was previously thevehicle to deliver the antibiotic ciprofloxacin with CPEs to the TM (8).For 18% [P407], the storage (G′) and loss (G″) moduli (measured bylinear oscillatory shear rheology at 100 rads⁻¹, 1% strain, 1° C. min⁻¹)were ˜1 kPa at room temperature; it behaved as a viscous liquid. G′ andG″ demonstrated sharp increases at temperatures above 27° C., andplateaued at ˜6 kPa and 4 kPa respectively (FIG. 13A), demonstratingsolid-like behavior. However, when 3CPE was added to the P407 solutionat the desired concentrations (which was previously used to enhancepermeation across the TM (8)), storage and loss moduli of theformulation were less than 2 kPa over the temperature range of 20-40° C.(FIG. 13B), i.e. the material did not form a gel in the presence of3CPE.

P407-PBP had faster gelation kinetics than did unmodified P407. Thesol-gel transition occurred ˜7 seconds after Cip-3CPE-18% [P407-PBP] wassubmerged into a water bath at 37° C., while a Cip-3CPE-18% [P407]remained as a solution for 48 seconds. The Cip-18% [P407] and Cip-18%[P407-PBP] (in the absence of 3CPE) demonstrated identical gelationkinetics.

Gelation of P407-PBP was not hindered by the inclusion of 3CPE. Linearoscillatory shear rheology measurements (100 rads⁻¹, 1% strain, 1° C.min⁻¹) indicated that the storage (G′) and loss (G″) moduli of Cip-18%[P407-PBP] were ˜0.3 and 1.0 kPa respectively at room temperature (FIG.13C). G′ and G″ both increased gradually in the temperature range 27-38°C. and became 7.8 and 5.0 kPa respectively near body temperature. Thesol-gel transition temperature was ˜33° C. Introduction of 3CPE toCip-18% [P407-PBP] increased its storage modulus more than 2.5 fold(FIG. 13D). G′ and G″ increased from close to zero at room temperatureto 20 and 1.3 kPa respectively at 37° C. The polymer solution exhibiteda sol-gel transition temperature of 20° C. By TEM, micelles wereobserved at the low polymer concentrations (1%) required for thatimaging modality.

To evaluate the effect of individual CPEs on temperature-dependentmechanical properties, 1% SDS, 2% limonene or 0.5% bupivacaine wereadded separately to Cip-18% [P407-PBP] (FIGS. 15A and 15B). Limonenereduced the gelation temperature of Cip-18% [P407-PBP] by 14° C.,increased G′ by 10.5 kPa and decreased G″ by 4.1 kPa, such that theywere very similar to those for Cip-3CPE-18% [P407-PBP] (FIGS. 13D and15B), suggesting that the influences of CPEs are dominated by theeffects of limonene. SDS shifted the gelation temperature of Cip-18%[P407-PBP] lower by ˜1.5° C., but did not affect the plateau values ofG′ and G″. Bupivacaine had minimal effects on the mechanical propertiesand gelation temperature of Cip-18% [P407-PBP].Syringing of Cip-3CPE-18%[P407-PBP] and Cip-3CPE-15% [P407-PBP] was challenging. For example,Cip-3CPE-15% [P407-PBP] was hard to push through a 20 gauge catheter andextruded as a gel rather than a viscous liquid, which made placement onthe TM in vivo difficult. Therefore, for in vivo work, Cip-3CPE-12%[P407-PBP] was selected, because its sol-gel transition was clear (G′>G″at body temperature; FIGS. 15C and 15D), and it did not gel afterextrusion through a 20 gauge, 1.8 inch catheter at room temperature.

In Vitro Drug Release and Ex Vivo Drug Flux

The design of the formulation entailed two components: CPEs that areexpected to increase drug flux across the TM (8), and the hydrogel,which is expected to slow flux but will prolong treatment for theduration that is needed for clearance of infection. The effect of thehydrogel and 3CPE on the transport rate of ciprofloxacin were studied byquantifying 1) in vitro diffusion from the bulk hydrogel matrix (albeitin infinite sink conditions, which are unlikely to exist on the TMsurface); 2) ex vivo permeation across the TM. In vitro releaseexperiments showed that P407-PBP slowed drug release compared to thefree drug solution (FIG. 19). Incorporation of 3CPE caused furtherslowing of drug release. However, the magnitude of ciprofloxacin releasefrom Cip-3CPE-18% [P407-PBP] was more than 30% greater than fromCip-3CPE-18% [P407]. Drug transport across the TM was studied ex vivo inauditory bullae excised from healthy chinchillas, as described inMethods. Inclusion of 3CPE enhanced the flux across the TM from a 1%ciprofloxacin solution more than 4 fold and from Cip-18% [P407-PBP] morethan 10 fold (FIG. 16). Conversely, the presence of a hydrogel tended todecrease flux across the TM; that effect could be overcome by theincorporation of CPEs. The ex vivo model could not be used todemonstrate the principal utility of the hydrogel, which is to prolongthe duration of drug flux across the TM by creating a stable depotsystem, because at 37° C. the TMs degraded after ˜48 hours due tomicrobial growth.

Tissue toxicity was a potentially important consideration since CPEs candisrupt the stratum corneum (28). The specific combination of 3CPE werechosen because it effectively enhances permeation and is non-toxic invivo (See 8; Simons et al., Chemical penetration enhancers and in situforming reservoirs for trans-tympanic drug delivery: progress towardimproved treatment of Otitis media. (Massachusetts Institute ofTechnology) (2008)). Cytotoxicity of the polymer and 3CPE was evaluatedwith a3-(4,5-dimethyl-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium,inner salt (MTS) assay in three cell lines that are representative ofcell types in the auditory system: human dermal fibroblasts (hFB), PC12cells (a pheochromocytoma cell line frequently used to testneurotoxicity), and normal adult human primary epidermal keratinocytesfrom abdominal skin (FIG. 20A). A LIVE/DEAD® Viability/Cytotoxicity Kitwas used as a confirmatory assay in hFB (FIG. 20B). Test formulationswere placed in the upper chamber of a Transwell system with a pore sizeof 0.4 μm, with cells below. P407-PBP itself showed little toxicityafter 1 day, but considerably more on day 3. The presence of 3CPE andciprofloxacin increased cytotoxicity for all cell types and time points.Nevertheless, biocompatibility was excellent in vivo: TMs treated with200 μL Cip-3CPE-18% [P407-PBP] were histologically similar to healthyTMs that had not been exposed to any treatment (FIG. 17A). Gels wereadherent to the TMs 7 days after application, but had degradedcompletely within 3 weeks.

Performance in Otitis Media In Vivo

OM due to NTHi was established in chinchillas following directinoculation into the middle ear, which were then treated with 200 μL oftest material deposited through the external canal on the TM. In animalstreated with 1% ciprofloxacin alone (n=8), NTHi were detectable in themiddle ear fluid of 25% animals on days 1 and 3, but by day 7 only in62.5% animals had infection had been cleared (FIG. 18A; i.e., zero cfu[colony-forming unit] in middle ear fluid aspirated from the dorsalaspect of the auditory bullae, i.e., not through the TM), anunacceptably low cure rate (31). The clearance rate was similarly low,60%, in animals with OM treated with Cip-3CPE-18% [P407] (n=5). Incontrast, OM was cleared in 10 of 10 animals treated with Cip-3CPE-12%[P407-PBP] (FIG. 18A; p=0.0065 by Fisher's exact test), within 24 hoursfollowing application of the formulation.

The 100% cure rate in animals treated with Cip-3CPE-12% [P407-PBP] maybe explained by the time course of ciprofloxacin levels in the middleear (FIG. 18B). The concentration of ciprofloxacin peaked at 1 day (39.1μg mL⁻¹ in animals treated with Cip-3CPE-12% [P407-PBP] and 4.2 μg mL⁻¹in those treated with 1% ciprofloxacin solution). Three days afteradministration of the formulations, ciprofloxacin was stillsupra-therapeutic (3.06 μg mL⁻¹) in the middle ears of animals thatreceived Cip-3CPE-12% [P407-PBP], while the ciprofloxacin concentrationdropped to zero in animals treated with 1% ciprofloxacin solution. (Theminimum inhibitory concentration (MIC) of NTHi by ciprofloxacin is0.1-0.5 μg mL⁻¹ (See Perez-Vazquez, et al., Antimicrob. AgentsChemother. 47, 3539-3541 (2003); Hirakata et al., Antimicrob. AgentsChemother. 53, 4225-4230 (2009)). Seven days after administration, theciprofloxacin concentration was still 1.2 μg mL⁻¹ in the middle earfluid of animals treated with Cip-3CPE-12% [P407-PBP], i.e. theconcentration of antibiotics in the auditory bullae was above MICthroughout a 7-day period in that group. No recurrence of OM wasobserved.

The fact that ciprofloxacin drops had any effectiveness at all in OM wassurprising, given that the TM is relatively impermeable to smallmolecules (such as CO₂ and He) (12, 14, 34) and ototopical antibioticsare only used for middle ear disease in situations where the TM has beenbreached (e.g. with myringotomy tubes) (Wall et al., Pediatr. Infect.Dis. J. 28, 141-144 (2009)). The effect of iprofloxacin drops in OM maybe explained by the fact that the TM became 5- to 31-fold more permeableto drug flux in OM (FIG. 16) despite also becoming much thicker (FIG.17A).

Systemic Distribution of Ciprofloxacin

Ciprofloxacin was undetectable in plasma samples from blood obtained inthe transverse sinus (table S2). Given the proximity of that location tothe auditory bullae, the absence of ciprofloxacin suggested that nosystemic exposure of antibiotics occurred.

Histological Assessment

The single-dose treatment with Cip-3CPE-12% [P407-PBP] was able toreverse the prominent inflammatory response caused by NTHi and toprevent bacterial growth (FIG. 17B). Seven days after administration ofthe formulation, the TM was excised intact within the tympanic ring, andprocessed into hematoxylin and eosin (H&E) stained sections. Normalchinchilla TMs were consistently 10-20 μm thick (FIG. 17A). TMsextracted after 7 days of infection were approximately five timesthicker (FIG. 17A) and exhibited an acute inflammatory response withdiffuse edema and dense infiltration by inflammatory cells. Incomparison, TMs treated with the gel formulation appearedindistinguishable from healthy TMs with a thickness of 10-20 μm (FIG.17A). No tissue injury, necrosis or inflammatory cells were observed.These results illustrate a benign tissue response to Cip-3CPE-12%[P407-PBP].

Effect of Cip-3CPE-12% [P407-PBP] on Hearing

The effect of Cip-3CPE-12% [P407-PBP] on hearing sensitivity wasassessed by auditory brainstem responses (ABR; see Methods; FIG. 18C).Placement of 200 μL of the gel on the TM caused a 16-24 dB positiveshift of the ABR threshold (worsening of hearing) to clicks and tonebursts of frequencies from 0.5 Hz to 16 kHz, with an average of 18 dB±8dB across all frequencies. This mild hearing loss is comparable to theeffect of cerumen (See Olusanya, et al., Ann. Trop. Paediatr. 23,121-128 (2003); Akinpelu et al., Int. J. Pediatr. Otorhi. 78, 88-90(2014)).

Gel Properties of a P407-PPE Polymer

Compositions for testing were prepared by mixing 18% wt/vol P407-PBPpolymer into an aqueous solution of 1% wt/vol ciprofloxacin. Thesolutions were stirred overnight and the following CPEs were added: (a)1% wt/vol sodium dodecylsulfate (SDS); (b) 2% wt/vol limonene (LIM); (c)0.5% wt/vol bupivacaine (Bup); or (d) 1% wt/vol SDS, 2% wt/vol LIM, and0.5% wt/vol Bup (3CPE).

Changes in mechanical properties during sol-gel transition werequantified using linear oscillatory shear rheology measurements (100rads⁻¹, 1% strain, 1° C. min⁻¹). As shown in FIG. 5. The storage (G′)and loss (G″) moduli of 18% P407 aqueous solution are around 0.1 kPa atroom temperature, both demonstrate sharp increases in the temperaturerange of 25-30° C., and then plateau around 6 kPa and 4 kParespectively. At temperatures below 27° C., the G′ and G″ remain closewithin one standard deviation (as indicated by the error bars) and P407behaves as a viscous liquid, whereas at temperatures above 28° C., G′becomes significantly larger than G″, and P407 demonstrates solid-likebehavior. Inclusion of CPEs (3CPE) in the P407 solution diminishes thegelation process and renders the delivery system ineffective.Specifically, Storage and loss moduli of the system remain less than 2kPa in the entire temperature range of 20-40° C. No cross-over point isobserved. The shear rheology results are consistent with our finding onotoscopy that the P407-based gels were spread out in the auditory canal,leading to poor adherence to the TM. The gelation process took ˜20 sec.

Rheology measurements for P407-PBP compositions are shown in FIG. 10.Linear oscillatory shear rheology measurements (100 rads⁻¹, 1% strain,1° C. min⁻¹) on 18% P407-PBP indicate that the storage (G′) and loss(G″) moduli of P407-PBP (containing 10 mg m1⁻¹ ciprofloxacin), arearound 0.1 and 0.2 kPa respectively under room temperature. G′ and G″both increase gradually within the temperature range of 27-38° C. andplateau at 10 and 4 kPa respectively. At temperatures below 33° C., G″is greater than G′ and the polymer solution behaves as a viscous liquid,whereas at temperatures above 33° C., G″ is smaller than G′, indicatingsolid-like behavior. The cross-over point (33° C.) is thus the sol-geltransition temperature. Introduction of CPEs, with the composition of 1wt % SDS, 2 wt % limonene and 0.5 wt % bupivacaine, changes the rheologyof P407-PBP. Upon increasing temperature, G′ and G″ increase from closeto zero to 10 and 5 kPa respectively at 22° C., and exhibit a cross-overpoint (i.e., sol-gel transition) at 20° C. G′ continues to increase withtemperature and plateaus at 20 kPa at body temperature, whereas G″starts to decrease with temperatures higher than 22° C. and plateaus at1 kPa at body temperature. Additional properties of the P407-PBPcompositions with various permeation enhancers are presented in TableE1.

TABLE E1 Rheological properties of exemplary P407-PBP compositions.Phase Transition Plateau storage Pleateau loss Temperature modulusmodulus CPE (° C.) (kPa) (kPa) no CPEs 33 9.7 4.9 0.5% bupivacaine 3310.8 4.9 1% sodium 31 10.2 4.8 dodecylsulfate 2% limonene 20 15.1 4.43CPE 20 19.1 5.3

Optimization of Formulations

The standard formulation is defined as ciprofloxacin in 18% P407 with 1%SDS, 0.5% bupivacaine, and 2% limonene. Starting from that formulation,others may be optimized with respect to gelation and mechanicalproperties, and drug flux across chinchilla TMs.

For example, an optimized formulation should produce a drug flux thatresults in a concentration in the recipient chamber of at least theminimum inhibitory concentration (MIC; the concentration that inhibitsthe growth of bacteria by 2 log units) within 12 hours. The MICs ofciprofloxacin are <0.1-0.5 for non-typable H. influenzae (NTHi) and0.5-4 μg/mL for S. pneumoniae. [31,32] For an optimized formulation,gelation should occur 10 sec after application while being fluid at roomtemperature, and should provide a drug flux that achieves MIC every dayfor 10 days. In vivo the optimized formulation should eradicateinfection in 100% of animals 5 days after treatment.

For optimization two CPEs (differing in carbon chain length) may beanalyzed from each of three principal classes: anionic, cationic, andnonionic (Table 1). Other CPEs that may be included in optimizationexperiments are: terpenes (e.g. limonene), benzalkonium chloride (anantiseptic and preservative used in eye drops and nasal sprays, alsoacts as a CPE), and bupivacaine (a potent local anesthetic, also acts asa CPE). Bupivicaine may also serve as an additional therapeutic agent totreat pain from OM.

TABLE E2 Properties of surfactant chemical penetration enhancers (CPEs).Length of CPE Class M.W. carbon chains Sodium octyl sulfate anionic 2328 Sodium dodecyl anionic 288 12 sulfate Octyl-trimethyl- cationic 252 8ammonium bromide Dodecyl-trimethyl- cationic 308 12 ammonium bromideTween 20 nonionic 1228 12 Tween 80 nonionic 1310 17

The antibiotic may be selected based on clinical criteria (antimicrobialspectrum, current practice; i.e. translatability), potency, solubilityin the delivery vehicle, stability at 37° C. and other physicochemicalparameters. The default antibiotic is ciprofloxacin because (a) it issmall (331 Da), moderately hydrophobic (log P=0.28), can be dissolved atrelatively high concentration in aqueous solution at acidic pH(pK_(a)=6.16), and has a broad antibacterial spectrum, (b) it iscurrently used clinically to treat acute otorrhea in children withtympanostomy tubes.

To minimize animal experimentation, chinchilla TMs can be used only forCPEs that achieve adequate flux in initial screening with cadaverichuman skin (HES). Since flux across TM is likely to be greater thanacross HES, the screen may also increases the probability thatformulations will be successful downstream in in vivo models of OM. Theintactness of human cadaveric skin and chinchilla TM samples can bedemonstrated by electrical impedance measurements. HES can be tested inFranz diffusion cells; chinchilla TMs in 12-well plates. For each drug,flux is to be measured at the maximum concentration that can bedissolved in the formulation. Flux of drug or CPE can be measured byHPLC with suitable detection.

Single CPEs

For each CPE, ciprofloxacin flux can be measured across HES, measuringflux at a range of concentrations starting with half of theconcentration shown to be effective in transdermal applications, [26a]and increasing by the same increment (or a multiple thereof) untiladequate concentrations are reached. The results for promising CPEs inHES test can be confirmed in chinchilla TMs prior to additionalexperiments. The experiments can be repeated with different therapeuticagents other than ciprofloxacin.

Synergistic CPEs.

Synergism between CPEs may be demonstrated formally by isobolographicanalysis (FIG. 6). For the two single enhancers that produce thegreatest increase in flux, the concentrations of both that causes 50% ofthe maximal increase in flux (EC₅₀) will be determined. If both are fromthe same class of enhancer, the next best agent from another class willalso be tested, since synergism is often found with processes that acton a common phenomenon by different mechanisms. Synergism (as well asadditivity and antagonism) can then be demonstrated by constructing anisobologram (FIG. 6). EC₅₀ values can be determined by logit (logisticregression) analysis, using Stata software (Stata Corporation, CollegeStation, Tex.).

An anesthetic permeation enhancer can boost the enhancement of drug fluxfor surfactant and terpene permeation enhancers. For example,bupivacaine can boost the enhancement of drug flux of SDS and Limonene(See FIG. 23).

Antibiotic Flux

In vivo studies with may initially be performed with ciprofloxacin.However, other antibiotics can also be studied to assess trans-tympanicdrug diffusion as a function of drug properties. Antibiotics that arecommonly used to treat otitis media may be studied, or that could beused to treat OM if systemic distribution and toxicity associated withoral delivery were not an issue. The target organisms includeStreptococcus pneumoniae, Haemophilus influenzae, and Moraxellacatarrhalis. Criteria to assess for a successful candidate drug includesolubility, stability, physicochemical properties, potency, and systemictoxicity. The properties of the TM are also likely to affect which drugswill work best. Candidates after ciprofloxacin include other quinoloneswith better Gram-positive coverage, greater potency, or less proteinbinding (e.g., levofloxacin and moxifloxacin) or broad-spectrum agentslike the carbapenems (e.g., meropenem). Drugs with pronouncedototoxicity (e.g. vancomycin) will not be studied.

For the antibiotic levoflaxin, the trans-tympanic permeation oflevofloxacin formulations (1.5% Levofloxacin aqueous solution and 1.5%Levofloxacin with permeation enhancers and a matrix forming agent) isshown in comparison with ciprofloxacin formulations in FIG. 23(B).

A panel of antibiotics is listed in Table 2, has been selected for arange of physicochemical properties. The flux of additional therapeuticagents (a) dexamethasone, which is used clinically in conjunction withantibiotics, and (b) β-lactamase inhibitors such as clavulanate andtazobactam, can also be investigated in combination with antibioticcandidates.

TABLE E3 Properties of antibiotics for use as therapeutic agents of thecomposition Antibiotic Class M.W. Log P Amoxicillin penicillin 365 0.87Azithromycin macrolide 749 4.02 Cefuroxime 2^(nd) generation 424 −0.16cephalosporin Ceftriaxone 3^(rd) generation 555 −1.47 cephalosporinTrimethoprim diaminopyrimidine 290 0.91 Ciprofloxacin quinolone 331 0.28

More than one antibiotic used in combination may also be tested if asingle antibiotic provides inadequate flux or fails to achieve MIC. Drugcombinations which are synergistic may allow increased flux ofantibacterial efficacy (peak effect) for a given total drug mass.Synergism can be investigated with the same statistical methodology asfor CPEs.

Encapsulation of Bupivacaine

Bupivacaine differs from the other CPEs in that it has a solid(free-base) form. This provides an opportunity to extend the duration ofCPE-effect (if needed) by sustained release from the drug deliverycomposition. Particles releasing bupivacaine can be suspended withinformulation. Experiments to verify that bupivacaine levels do not riseto neurotoxic levels in the middle or inner ear may be necessary.

Measurement of Drug Flux Across Human Skin

Heat-stripped epidermis with stratum corneum (HES) can be prepared fromfresh frozen, full-thickness, hairless human abdominal skin (NationalDisease Research Interchange, Philadelphia, Pa.). [38] HES is securedbetween the orifices of vertical (Franz) diffusion cells (Permegear,Bethlehem, Pa.). At fixed time points, samples are removed from thereceiving chamber and analyzed by HPLC.

Ex Vivo Measurement of Flux with Tympanic Membranes

The external auditory meatus and TM within the tympanic ring areseparated en bloc from the skull. This bloc (which will act as the donorchamber) is placed into 12-well plates and pre-incubated at 37° C. for15 minutes. 200 μL of a test solution is added to the donor chamber. Atfixed time points, receiving medium are removed. Drug concentrations arequantified by reverse-phase HPLC (1100 series, Agilent Technologies,Palo Alto, Calif.).

Determination of Intactness of HES and TMs

Intactness of skin and TM samples can be assessed with electricalimpedance measurements. [39] Skin samples and TMs with initialresistivities (electrical resistance*exposed area) of <35 kOhm*cm² and<18 kOhm*cm² respectively are considered damaged.

Hydrogel Formulation

Hydrogels can be prepared by adding polymer powders to aqueous drug-CPEsolutions. Gels of varying P407-PPE (co-poloxamer 407/polyphosphoester)weight percentages (5%-20%) are prepared by simple dissolution. In situcovalently cross-linking polymers (1-10 weight %) can be synthesized,[25] dissolved in antibiotic-CPE solution, and delivered in separatebarrels of a doubled-barreled syringe.

Example of Synthesis of a P407-PPE Polymer

Phosphate ester precursors (e.g., compound of Formula (A)) can beprepared by condensation reaction of2-chloro-2-oxo-1,3,2-dioxaphospholane (COP) and an alcohol (e.g., Y—OH,wherein Y is a defined herein), then purified by vacuum distillation,and analyzed by proton and phosphorous NMR spectroscopy. HydrophobicP407-PPE polymer (PPE-P407-PPE) can be synthesized by ring openingpolymerization (ROP) of the phosphate ester with P407 in the presence ofan organocatalyst, 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) at −20° C.Upon completion of the reaction (complete monomer consumption confirmedby NMR spectroscopy), excess acetic acid in dichloromethane (DCM) can beadded to the reaction mixture to quench the reaction. The product may bepurified by precipitation into ether (3 times) and dried to a whitepowder under vacuum. Proton and phosphorous NMR spectroscopy, Fouriertransform infrared spectroscopy and gel permeation chromatography areused to characterize the polymer and confirm its purity.

Gelation Temperature and Time, Gel Rheology

The storage and loss moduli can be measured every 1° C. during atemperature sweep from 0° C. to 40° C. The temperature at which thestorage modulus exceeds the loss modulus is considered the gelationtemperature. To measure gelation time, formulations in scintillationvials will be immersed in a 37° C. water bath over a stir plate. Thetime it takes for the stir bar to stop rotating is noted as the gelationtime.

In Vitro Release Kinetics

Release of drug and/or CPE from formulations can be assessed by placingthe gels in low molecular-weight cut-off (Transwell) inserts in 12-wellplates, with PBS below. At fixed time points (0.5, 1, 2, 6, 24, 48, 120h), samples of PBS from the receiving chamber are removed and analyzedby HPLC or other analytical technique for drug and/or CPE levels.

Cytotoxicity Testing

Cytotoxicity towards cell types that occur in the tympanic membrane andthe surrounding walls of the outer ear can be determined. These celltypes include keratinocytes, fibroblasts and PC12 cells (apheochromocytoma cell line often used to study neurotoxicity). Cells areexposed to a range of concentrations of drugs, CPEs, and gel components.For the CPEs, the initial upper concentration limit is set by publishedvalues for skin toxicity. For the drugs, the upper limit is set bysolubility in the formulations to be tested. Cytotoxicity is assessed at1 to 10 days of exposure to the component(s) being tested, using the MTTassay, which is widely used for cytotoxicity screening. Since it canalso reflect cell proliferation, a standard live-dead assay will be usedas a confirmatory test. [50]

Biocompatibility Testing

Formulations that show over 80% cell survival will be tested in vivo.Under isoflurane:oxygen anesthesia, 200 μL of test solutions isinstilled onto the chinchilla TM. One, four, ten and thirty days later,animals are euthanized for otoscopy and histological analysis of the TMand outer ear, with attention to material residue (and its adherence tothe TM), inflammation, thickening of the TM, middle ear effusion, andtissue injury. The time points will allow analysis of how longformulations last in the auditory canal. Dissection will proceed as forremoving TM's, but the outer and middle ear will be removed en bloc anddemineralized for subsequent sectioning, and processed intohematoxylin-eosin stained sections using standard procedures. Electronmicroscopy of inner ear structures may also be performed to assessototoxicity.

Biofilms

An in vitro study of the effects of the formulation components onbiofilm formation may be performed as an adjunct to the observations tobe obtained in the in vivo model. Formed biofilms can be exposed toconcentrations corresponding to dose-response curves of all thediffusible components of the formulations (drug, CPE, hydrogelprecursors), alone and in combination, and assessed for changes inmorphology and bacterial population. Analogous studies can be done toassess the components' ability to prevent biofilm formation in vitro,and to destroy devitalized biofilms.

Bacterial colonies are suspended in media and the OD₄₉₀ adjusted to0.65, then diluted 1:6 and incubated at 37° C. with 5% CO₂ forapproximately 3 hours in order to reach mid-log phase. [30] Thesuspension is then diluted 1:2500 with media and 200 μL placed into eachwell of an 8-well chamber slide and incubated at 37° C. with 5% CO₂ forapproximately 16 hours. The medium is changed every 12 hours, withattention to not disrupt the biofilm, until a desirable biofilmthickness is achieved. Samples are then fixed and stained with alive-dead assay. Biofilm thickness and bacterial survival can bequantitated by confocal microscopy, and further characterized with SEMimage and/or immunohistochemical approaches.

In Vivo Chinchilla Testing

To determine the efficacy of the antibacterial hydrogel in vivo,formulations can be applied to the TM's of chinchillas with OM. Prior tobacterial challenge, chinchillas are examined by tympanometry andotomicroscopy to confirm normal middle ear and TM status. Animals willhave a test composition placed in the left ear. The right ear is usedfor controls (no treatment, CPE only, gel only, etc.). In selectexperiments, middle ear fluid may be sampled to track bactericidaleffect and flux of antibiotics and CPEs.

Animals will be challenged by direct inoculation of 25-100 cfu into themiddle ear through the superior bullae. After 48-96 hours, infection isconfirmed by (a) otoscopy and tympanometry, (b) culture of the middleear fluid through a 3-5-mm opening in the bulla bone (made underketamine/xylazine anesthesia); results come back overnight. In ourexperience, virtually all animals develop disease after directinoculation. In the event that an animal does not develop disease, itwill be excluded from the study. Once the presence of otitis media isconfirmed hydrogels will be applied to chinchillas lying on their sides,under ketamine/xylazine anesthesia.

To assess biofilm, the middle ear mucosa will be visualized, [52] andtissue samples from animals analyzed by scanning electron microscopy(SEM) to detect biofilm and live-dead staining to detect viable bacteriawithin. [21] Immunohistochemistry for bacteria may be used as aconfirmatory test. These data will allow for the determination of theeffect of the various experimental groups on biofilm formation. Theeffect of CPEs without antibiotics on biofilm formation in OM can alsobe studied.

For studies of prophylaxis, a strategy for induction of experimentalotitis media designed to mimic the pathogenesis of disease in childrenmay be used, where colonization followed by viral respiratory tractinfection leading to negative middle ear pressure is observed. 10⁷-10⁸cfu of bacteria is inoculated into the nasopharynx of chinchillas usinga small gauge angiocatheter. After 24 hours, nasopharyngeal colonizationis confirmed by quantitative culture. [29 g, 51] Gel is placed in theleft external canal (in contact with the TM). Forty eight hours aftergel application barotrauma is introduced by placing a 25 gauge needle inthe middle ear (through the superior bullae) and withdrawing of 500 μLof air while anesthetized tympanometry is performed to document thepresence of negative middle ear pressure within the middle ear cavity.This creates negative pressure that remains for several hours andinduces bacterial otopathogens to ascend the Eustachian tube into themiddle ear. Animals are observed daily for the development of OM and ifchanges in TM are observed, culture is performed. (If no changes areobserved culture will be performed 3-4 days after barotrauma to confirmthe absence of culture positive disease).

In both paradigms, 0.2 mL of test composition (hydrogel with drug andCPE) is applied onto the TM through a syringe with an attachedangiocatheter, under otoscopic observation. The entire surface of the TMis coated. Clinical examinations take place as above and/or 1, 3, 5, and7 days after drug administration to monitor disease. Otoscopy will beused to follow contact of the hydrogel with the TM. Every other day,middle ear fluid, if present, is collected via an angiocatheter insertedthrough the incision made during initial culture confirmation underaseptic conditions. In the absence of middle ear fluid, lavage will beperformed with 500 μL of Hanks solution and aspiration through anangiocatheter. Quantitative middle ear fluid cultures are performed by10 fold dilution of the middle ear fluid and incubation at 37° C. for 16hours.

Drug levels in the middle ear can be determined by (a) addition ofmethanol to middle ear fluid until all protein is precipitated, (b)centrifugation to remove any precipitated protein and cellular debris,and (c) analysis by HPLC.

Less than 2 mL of blood can be collected by superior sagittal sinuspuncture at specified intervals after initiating treatment to measuresystemic (plasma) drug concentrations. Blood (≤2 mL) will be drawn fromanimals that have had formulations deposited in their ears forbiocompatibility testing or the OM models, by superior sagittal sinuspuncture, placed on ice immediately, and plasma separated bycentrifugation. Samples will be stored at −20° C. and antibiotic and/orCPE concentrations subsequently measured. The levels after days one,four, and ten provide useful survey of values over the course oftreatment.

Acoustic Brain Response

Impairment of hearing could be caused by a conductive effect of thegels, or by direct toxicity to the middle or inner ear. It is difficulta priori to predict the thickness of the formulation that will beapplied in an eventual therapeutic system in humans. A range ofthicknesses from 100 μm to 500 μm will be applied, which fills theauditory canal of the chinchilla completely, prior to measurement of theacoustic brain response (ABR). To identify possible ototoxic effects,testing will be repeated after removing the gels (by rinsing and/orcurettage, depending on the consistency).

ABR experiments will be conducted with a custom-designed system builtaround National Instruments (Austin, Tex.) software (Lab View) andhardware including a GPIB controller and an ADC board. The customLabView program computes the stimuli, and downloads them to aprogrammable stimulus generator (Hewlett Packard 33120A). The stimulusis then filtered by an antialiasing filter (KrohnHite 3901) andattenuated (Tucker-Davis Technologies). Simultaneously with stimulusoutput, the 2 ADC channels sample the amplified ABR signal and theoutput of a microphone sealed in the ear canal of the animal.

The acoustic stimuli will be pairs of 20-ms tone bursts of oppositepolarity. The frequency of the bursts will increase from 500 Hz to 16kHz in octave steps. Each burst will be sine windowed, with 40 msbetween two bursts. ABR responses to 250 pairs of stimuli will beaveraged at each stimulus level. The ABR response will be computed fromthe sum of the averaged response to the two different polarities.Stimulus level will be varied in 10 dB steps. A visual judgment ofthreshold at each stimulus frequency will be determined post-measurementin a blinded fashion.

The attenuated stimulus will be played through a hearing-aid earphoneplaced within the intact ear canal of adult male chinchillas (400-600 g)anesthetized by IP administration of ketamine and pentobarbital (50mg/kg). The earphone coupler includes a microphone that monitors thesound stimulus level. ABRs, obtained in a sound-attenuating booth, willbe measured with a differential amplifier with a gain of 10,000 and ameasurement bandwidth of 100 Hz to 3 kHz. The measurements will beobtained from the positive electrode in the muscle behind the measuredear; the negative electrode will be at the cranial vertex, and theground electrode behind the contralateral ear.

REFERENCES

-   1. (a) Berman, S., Otitis media in children. N Engl J Med 1995, 332,    1560-5; (b) Fried, V. M.; Makuc, D. M.; Rooks, R. N. Ambulatory    health care visits by children: principal diagnosis and place of    visit.; 137; Washington, D.C.: Government Printing Office, 1998.:    1998.-   2. Teele, D. W.; Klein, J. O.; Rosner, B., Epidemiology of otitis    media during the first seven years of life in children in greater    Boston: a prospective, cohort study. The Journal of infectious    diseases 1989, 160 (1), 83-94.-   3. Casselbrant, M. L.; Mandel, E. M., Epidemiology. In    Evidence-based otitis media, Rosenfeld, R. M.; Bluestone, C. D.,    Eds. Decker, Inc.: Hamilton, British Columbia, 1999; pp 117-137.-   4. Faden, H.; Duffy, L.; Boeve, M., Otitis media: back to basics.    The Pediatric infectious disease journal 1998, 17 (12), 1105-12;    quiz 1112-3.-   5. Lanphear, B. P.; Byrd, R. S.; Auinger, P.; Hall, C. B.,    Increasing prevalence of recurrent otitis media among children in    the United States. Pediatrics 1997, 99 (3), E1.-   6. Acuin, J. Otitis Media: Burden of Illness and Management Options;    World Health Organization: Geneva, Switzerland, 2004.-   7. (a) Bluestone, C. D.; Klein, J. O., Otitis media in infants and    children. 4th ed.; BC Decker: Hamilton, Ontario, Canada, 2006; (b)    Bluestone, C. D.; Klein, J. O., Otitis media in infants and    children. BC Decker: Hamilton, O N, 2007.-   8. Khoo, X.; Simons, E.; Chiang, H.; Hickey, J.; Sabharwal, V.;    Pelton, S.; Rosowski, J.; Langer, R.; Kohane, D., Formulations for    trans-tympanic antibiotic delivery. Biomaterials 2013, 34, 1281-8.-   9. Paradise, J. L., Short-course antimicrobial treatment for acute    otitis media: not best for infants and young children. Jama 1997,    278 (20), 1640-2.-   10. Antibiotic/Antimicrobial Resistance.    www.cdc.gov/drugresistance/.-   11. Doyle, W. J.; Alper, C. M.; Seroky, J. T.; Karnavas, W. J.,    Exchange rates of gases across the tympanic membrane in rhesus    monkeys. Acta oto-laryngologica 1998, 118 (4), 567-73.-   12. Suzuki, K.; Baba, S., Antimicrobial ear drop medication therapy.    Acta Otolaryngol Suppl 1996, 525, 68-72.-   13. Middleton, J. D., Mechanism of action of surfactants on water    binding properties of isolated stratum corneum. J Soc Cosmet Chem    1969, 20, 399-403.-   14. Kushla, G. P.; Zatz, J. L.; Mills, O. H., Jr.; Berger, R. S.,    Noninvasive assessment of anesthetic activity of topical lidocaine    formulations. J Pharm Sci 1993, 82 (11), 1118-22.-   15. Walker, R. B.; Smith, E. W., The role of percutaneous    penetration enhancers. Adv Drug Deliv Rev 1996, 18, 295-301.-   16. (a) Jia, X.; Colombo, G.; Padera, R.; Langer, R.; Kohane, D. S.,    Prolongation of sciatic nerve blockade by in situ cross-linked    hyaluronic acid. Biomaterials 2004, 25 (19), 4797-804; (b) Yeo, Y.;    Bellas, E.; Highley, C. B.; Langer, R.; Kohane, D. S., Peritoneal    adhesion prevention with an in situ cross-linkable hyaluronan gel    containing tissue-type plasminogen activator in a rabbit    repeated-injury model. Biomaterials 2007, 28, 3704-13; (c) Hoare,    T.; Kohane, D. S., Hydrogels in drug delivery: progress and    challenges. Polymer 2008, 49, 1993-2007.-   17. Yeo, Y.; Kohane, D. S., Polymers in the prevention of peritoneal    adhesions. Eur J Pharm Biopharm 2008, 68, 57-66.-   18. (a) Hall-Stoodley, L.; Hu, F. Z.; Gieseke, A.; Nistico, L.;    Nguyen, D.; Hayes, J.; Forbes, M.; Greenberg, D. P.; Dice, B.;    Burrows, A.; Wackym, P. A.; Stoodley, P.; Post, J. C.; Ehrlich, G.    D.; Kerschner, J. E., Direct detection of bacterial biofilms on the    middle-ear mucosa of children with chronic otitis media. Jama 2006,    296 (2), 202-11; (b) Post, J. C.; Hiller, N. L.; Nistico, L.;    Stoodley, P.; Ehrlich, G. D., The role of biofilms in    otolaryngologic infections: update 2007. Curr Opin Otolaryngol Head    Neck Surg 2007, 15 (5), 347-51; (c) Liu, Y. C.; Post, J. C.,    Biofilms in pediatric respiratory and related infections. Curr    Allergy Asthma Rep 2009, 9 (6), 449-55.-   19. Nistico, L.; Kreft, R.; Gieseke, A.; Coticchia, J. M.; Burrows,    A.; Khampang, P.; Liu, Y.; Kerschner, J. E.; Post, J. C.; Lonergan,    S.; Sampath, R.; Hu, F. Z.; Ehrlich, G. D.; Stoodley, P.;    Hall-Stoodley, L., Adenoid reservoir for pathogenic biofilm    bacteria. J Clin Microbiol 2011, 49 (4), 1411-20.-   20. Hoa, M.; Syamal, M.; Sachdeva, L.; Berk, R.; Coticchia, J.,    Demonstration of nasopharyngeal and middle ear mucosal biofilms in    an animal model of acute otitis media. Ann Otol Rhinol Laryngol    2009, 118 (4), 292-8.-   21. (a) Hoa, M.; Tomovic, S.; Nistico, L.; Hall-Stoodley, L.;    Stoodley, P.; Sachdeva, L.; Berk, R.; Coticchia, J. M.,    Identification of adenoid biofilms with middle ear pathogens in    otitis-prone children utilizing SEM and FISH. Int J Pediatr    Otorhinolaryngol 2009, 73 (9), 1242-8; (b) Lee, M. R.; Pawlowski, K.    S.; Luong, A.; Furze, A. D.; Roland, P. S., Biofilm presence in    humans with chronic suppurative otitis media. Otolaryngol Head Neck    Surg 2009, 141 (5), 567-71; (c) Hoa, M.; Syamal, M.; Schaeffer, M.    A.; Sachdeva, L.; Berk, R.; Coticchia, J., Biofilms and chronic    otitis media: an initial exploration into the role of biofilms in    the pathogenesis of chronic otitis media. Am J Otolaryngol 2010, 31    (4), 241-5.-   22. Tapiainen, T.; Kujala, T.; Kaijalainen, T.; Ikaheimo, I.;    Saukkoriipi, A.; Renko, M.; Salo, J.; Leinonen, M.; Uhari, M.,    Biofilm formation by Streptococcus pneumoniae isolates from    paediatric patients. Apmis 2010, 118 (4), 255-60.-   23. (a) Kohane, D. S.; Yieh, J.; Lu, N. T.; Langer, R.;    Strichartz, G. R.; Berde, C. B., A re-examination of tetrodotoxin    for prolonged duration local anesthesia. Anesthesiology 1998, 89    (1), 119-31; (b) Kohane, D. S.; Sankar, W. N.; Shubina, M.; Hu, D.;    Rifai, N.; Berde, C. B., Sciatic nerve blockade in infant,    adolescent, and adult rats: a comparison of ropivacaine with    bupivacaine. Anesthesiology 1998, 89 (5), 1199-208; (c) Kohane, D.    S.; Lu, N. T.; Gokgol-Kline, A. C.; Shubina, M.; Kuang, Y.; Hall,    S.; Strichartz, G. R.; Berde, C. B., The local anesthetic properties    and toxicity of saxitonin homologues for rat sciatic nerve block in    vivo. Reg Anesth Pain Med 2000, 25 (1), 52-9; (d) Kohane, D. S.;    Lu, N. T.; Crosa, G. A.; Kuang, Y.; Berde, C. B., High    concentrations of adrenergic antagonists prolong sciatic nerve    blockade by tetrodotoxin. Acta Anaesthesiol Scand 2001, 45 (7),    899-905; (e) Kohane, D. S.; Lu, N. T.; Cairns, B. E.; Berde, C. B.,    Effects of adrenergic agonists and antagonists on    tetrodotoxin-induced nerve block. Reg Anesth Pain Med 2001, 26 (3),    239-45; (f) Padera, R.; Bellas, E.; Tse, J. Y.; Hao, D. D.;    Kohane, D. S., Local myotoxicity from sustained release of    bupivacaine from microparticles. Anesthesiology 2008, 108, 921-8.-   24. Kohane, D. S.; Kuang, Y.; Lu, N. T.; Langer, R.; Strichartz, G.    R.; Berde, C. B., Vanilloid receptor agonists potentiate the in vivo    local anesthetic activity of percutaneously injected site 1 sodium    channel blockers. Anesthesiology 1999, 90, 524-534.-   25. (a) Ito, T.; Fraser, I. P.; Yeo, Y.; Highley, C. B.; Bellas, E.;    Kohane, D. S., Anti-inflammatory function of an in-situ    cross-linkable conjugate hydrogel of hyaluronic acid and    dexamethasone. Biomaterials 2007, 28 (10), 1778-1786; (b) Hudson, S.    P.; Langer, R.; Fink, G. R.; Kohane, D. S., Injectable in situ    cross-linking hydrogels for local antifungal therapy. Biomaterials    2010, 31, 1444-52; (c) Yeo, Y.; Adil, M.; Bellas, E.; Astashkhina,    A.; Chaudary, N.; Kohane, D. S., Prevention of peritoneal adhesions    with an in situ cross-linkable hyaluronan hydrogel delivering    budesonide. J Control Release 2007, 120, 178-85; (d) Hoare, T.;    Bellas, E.; Zurakowski, D.; Kohane, D. S., Rheological blends for    drug delivery. II: Prolongation of nerve blockade, biocompatibility,    and in vitro-in vivo correlations. J Biomed Mater Res A 2010, 92,    586-95; (e) Hoare, T.; Zurakowski, D.; Langer, R.; Kohane, D. S.,    Rheological blends for drug delivery. I: Characterization in vitro.    J Biomed Mater Res A 2010, 92, 575-85; (f) Chen, P. C.; Kohane, D.    S.; Park, Y. J.; Bartlett, R. H.; Langer, R.; Yang, V. C.,    Injectable microparticle-gel system for prolonged and localized    lidocaine release. II. In vivo anesthetic effects. J Biomed Mater    Res A 2004, 70 (3), 459-66; (g) Chen, P. C.; Park, Y. J.; Chang, L.    C.; Kohane, D. S.; Bartlett, R. H.; Langer, R.; Yang, V. C.,    Injectable microparticle-gel system for prolonged and localized    lidocaine release. I. In vitro characterization. J Biomed Mater Res    A 2004, 70 (3), 412-9; (h) Yeo, Y.; Bellas, E.; Firestone, W.;    Langer, R.; Kohane, D. S., Complex coacervates for thermally    sensitive controlled release of flavor compounds. J Agric Food Chem    2005, 53 (19), 7518-25; (i) Yeo, Y.; Burdick, J. A.; Highley, C. B.;    Marini, R.; Langer, R.; Kohane, D. S., Peritoneal application of    chitosan and UV-cross-linkable chitosan. J Biomed Mater Res A 2006,    78 (4), 668-75; (j) Yeo, Y.; Highley, C. B.; Bellas, E.; Ito, T.;    Marini, R.; Langer, R.; Kohane, D. S., In situ cross-linkable    hyaluronic acid hydrogels prevent post-operative abdominal adhesions    in a rabbit model. Biomaterials 2006, 27, 4698-4705; (k) Yeo, Y.;    Ito, T.; Bellas, E.; Highley, C. B.; Marini, R.; Kohane, D. S., In    situ cross-linkable hyaluronan hydrogels containing polymeric    nanoparticles for preventing post-surgical adhesions. Ann Surg 2007,    245, 819-824; (1) Ito, T.; Yeo, Y.; Highley, C. B.; Bellas, E.;    Benitez, C. A.; Kohane, D. S., The prevention of peritoneal    adhesions by in-situ cross-linking hydrogels of hyaluronic acid and    cellulose derivatives. Biomaterials 2007, 28 (6), 975-83; (m) Ito,    T.; Yeo, Y.; Highley, C. B.; Bellas, E.; Kohane, D. S.,    Dextran-based in situ cross-linked injectable hydrogels to prevent    peritoneal adhesions. Biomaterials 2007, 28, 3428-26; (n) Hoare, T.;    Yeo, Y.; Bellas, E.; Bruggeman, J. P.; Kohane, D. S., Prevention of    peritoneal adhesions using hyaluronic acid-hydroxypropylmethyl    cellulose rheological blends Acta biomaterialia 2014, 10, 1187-93.-   26. (a) Simons, E. J.; Bellas, E.; Lawlor, M. W.; Kohane, D. S.,    Effect of chemical permeation enhancers on nerve blockade. Mol    Pharmaceutics 2009, 6, 265-273; (b) Sagie, I.; Kohane, D. S.,    Prolonged sensory-selective nerve blockade. Proc Natl Acad Sci USA    2010, 107, 3740-5.-   27. (a) Zumbuehl, A.; Ferreira, L.; Kuhn, D.; Asthashkina, A.; Long,    L.; Yeo, Y.; Iaconis, T.; Ghannoum, M.; Fink, G. R.; Langer, R.;    Kohane, D. S., Antifungal hydrogels. Proc Natl Acad Sci USA 2007,    104, 12994-8; (b) Tsifansky, M. D.; Yeo, Y.; Evgenov, O. V.; Bellas,    E.; Benjamin, J.; Kohane, D. S., Microparticles for inhalational    delivery of antipseudomonal antibiotics. AAPS Journal 2008, 10,    254-60; (c) Ciolino, J. B.; Hoare, T. R.; Iwata, N. G.; Behlau, I.;    Dohlman, C. H.; Langer, R.; Kohane, D. S., A drug-eluting contact    lens. Invest Ophthalmol Vis Sci 2009, 50, 3346-42; (d) Ciolino, J.    B.; Hudson, S. P.; Mobbs, A. N.; Hoare, T. R.; Iwata, N.; Fink, G.    R.; Kohane, D. S., A prototype antifungal contact lens. Invest    Ophthalmol Vis Sci 2011, 52 (9), 6286-91; (e) Malavia, N.;    Zurakowski, D.; Schroeder, A.; Princiotto, A.; Laury, A.;    Epstein-Barash, H.; Sodroski, J.; Langer, R.; Madani, N.; Kohane, D.    S., Liposomes for HIV prophylaxis. Biomaterials 2011, 32 (33),    8663-8.-   28. Karande, P.; Jain, A.; Ergun, K.; Kispersky, V.; Mitragotri, S.,    Design principles of chemical penetration enhancers for transdermal    drug delivery. Proc Natl Acad Sci USA 2005, 102 (13), 4688-93.-   29. (a) Karasic, R. B.; Trumpp, C. E.; Gnehm, H. E.; Rice, P. A.;    Pelton, S. I., Modification of otitis media in chinchillas    rechallenged with nontypable Haemophilus influenzae and serological    response to outer membrane antigens. The Journal of infectious    diseases 1985, 151 (2), 273-9; (b) Pelton, S. I.; Figueira, M.;    Albut, R.; Stalker, D., Efficacy of linezolid in experimental otitis    media. Antimicrob Agents Chemother 2000, 44 (3), 654-7; (c) Babl, F.    E.; Pelton, S. I.; Li, Z., Experimental acute otitis media due to    nontypeable Haemophilus influenzae: comparison of high and low    azithromycin doses with placebo. Antimicrob Agents Chemother 2002,    46 (7), 2194-9; (d) Bouchet, V.; Hood, D. W.; Li, J.; Brisson, J.    R.; Randle, G. A.; Martin, A.; Li, Z.; Goldstein, R.; Schweda, E.    K.; Pelton, S. I.; Richards, J. C.; Moxon, E. R., Host-derived    sialic acid is incorporated into Haemophilus influenzae    lipopolysaccharide and is a major virulence factor in experimental    otitis media. Proc Natl Acad Sci USA 2003, 100 (15), 8898-903; (e)    Sabharwal, V.; Figueira, M.; Pelton, S. I.; Pettigrew, M. M.,    Virulence of Streptococcus pneumoniae serotype 6C in experimental    otitis media. Microbes Infect 2012, 14 (9), 712-8; (f) Sabharwal,    V.; Stevenson, A.; Figueira, M.; Orthopoulos, G.; Trzcinski, K.;    Pelton, S. I., Capsular switching as a strategy to increase    pneumococcal virulence in experimental otitis media model. Microbes    Infect 2014, 16 (4), 292-9; (g) Figueira, M.; Moschioni, M.; De    Angelis, G.; Barocchi, M.; Sabharwal, V.; Masignani, V.; Pelton, S.    I., Variation of pneumococcal Pilus-1 expression results in vaccine    escape during Experimental Otitis Media [EOM]. PLoS One 2014, 9 (1),    e83798.-   30. Jurcisek, J. A.; Dickson, A. C.; Bruggeman, M. E.; Bakaletz, L.    O., In vitro biofilm formation in an 8-well chamber slide. J Vis Exp    2011, (47).-   31. (a) Perez-Vazquez, M.; Roman, F.; Aracil, B.; Canton, R.;    Campos, J., In vitro activities of garenoxacin (BMS-284756) against    Haemophilus influenzae isolates with different fluoroquinolone    susceptibilities. Antimicrob Agents Chemother 2003, 47 (11),    3539-41; (b) Hirakata, Y.; Ohmori, K.; Mikuriya, M.; Saika, T.;    Matsuzaki, K.; Hasegawa, M.; Hatta, M.; Yamamoto, N.; Kunishima, H.;    Yano, H.; Kitagawa, M.; Arai, K.; Kawakami, K.; Kobayashi, I.;    Jones, R. N.; Kohno, S.; Yamaguchi, K.; Kaku, M., Antimicrobial    activities of piperacillin-tazobactam against Haemophilus influenzae    isolates, including beta-lactamase-negative ampicillin-resistant and    beta-lactamase-positive amoxicillin-clavulanate-resistant isolates,    and mutations in their quinolone resistance-determining regions.    Antimicrob Agents Chemother 2009, 53 (10), 4225-30.-   32. (a) Kayser, F. H.; Novak, J., In vitro activity of ciprofloxacin    against gram-positive bacteria. An overview. Am J Med 1987, 82 (4A),    33-9; (b) Patel, S. N.; McGeer, A.; Melano, R.; Tyrrell, G. J.;    Green, K.; Pillai, D. R.; Low, D. E., Susceptibility of    Streptococcus pneumoniae to fluoroquinolones in Canada. Antimicrob    Agents Chemother 2011, 55 (8), 3703-8.-   33. Jacobs, M. R., How can we predict bacterial eradication? Int J    Infect Dis 2003, 7 Suppl 1, S13-20.-   34. Barnet, C. S.; Tse, J. Y.; Kohane, D. S., Site 1 sodium channel    blockers prolong the duration of sciatic nerve blockade from    tricyclic antidepressants. Pain 2004, 110 (1-2), 432-8.-   35. Christodoulou, P.; Doxas, P. G.; Papadakis, C. E.; Prassopoulos,    P.; Maris, T.; Helidonis, E. S., Transtympanic iontophoresis of    gadopentetate dimeglumine: Preliminary results. Otolaryngol Head    Neck Surg 2003, 129 (4), 408-13.-   36. Bernards, C. M.; Hill, H. F., Physical and chemical properties    of drug molecules governing their diffusion through the spinal    meninges. Anesthesiology 1992, 77, 750-756.-   37. (a) Kohane, D. S.; Lipp, M.; Kinney, R. C.; Anthony, D. C.;    Louis, D. N.; Lotan, N.; Langer, R., Biocompatibility of    lipid-protein-sugar particles containing bupivacaine in the    epineurium. J Biomed Mater Res 2002, 59 (3), 450-9; (b) Kohane, D.    S.; Lipp, M.; Kinney, R. C.; Lotan, N.; Langer, R., Sciatic nerve    blockade with lipid-protein-sugar particles containing bupivacaine.    Pharm Res 2000, 17 (10), 1243-9; (c) Kohane, D. S.; Smith, S. E.;    Louis, D. N.; Colombo, G.; Ghoroghchian, P.; Hunfeld, N. G.;    Berde, C. B.; Langer, R., Prolonged duration local anesthesia from    tetrodotoxin-enhanced local anesthetic microspheres. Pain 2003, 104    (1-2), 415-21; (d) Colombo, G.; Langer, R.; Kohane, D. S., Effect of    excipient composition on the biocompatibility of    bupivacaine-containing microparticles at the sciatic nerve. J Biomed    Mater Res A 2004, 68 (4), 651-9; (e) Colombo, G.; Padera, R.;    Langer, R.; Kohane, D. S., Prolonged duration local anesthesia with    lipid-protein-sugar particles containing bupivacaine and    dexamethasone. J Biomed Mater Res A 2005, 75A (2), 458-464.-   38. Pliquett, U.; Prausnitz, M., Electrical Impedance Spectroscopy    for Rapid and Noninvasive Analysis of Skin Electroporation. In    Electrochemotherapy, Electrogenetherapy, and Transdermal Drug    Delivery, Jaroszeski, M.; Heller, R.; Gilbert, R., Eds. Humana    Press: 2000; Vol. 37, pp 377-406.-   39. Tang, H.; Mitragotri, S.; Blankschtein, D.; Langer, R.,    Theoretical description of transdermal transport of hydrophilic    permeants: application to low-frequency sonophoresis. J Pharm Sci    2001, 90 (5), 545-68.-   40. Kushner, J.; Blankschtein, D.; Langer, R., Experimental    demonstration of the existence of highly permeable localized    transport regions in low-frequency sonophoresis. J Pharm Sci 2004,    93 (11), 2733-45.-   41. Hecht, E.; Mortensen, K.; Gradzielski, M.; Hoffmann, H.,    Interaction of ABA block copolymers with ionic surfactants:    influence on micellization and gelation. The Journal of Physical    Chemistry 1995, 99 (13), 4866-4874.-   42. (a) Wetton, R. E.; Allen, G., The dynamic mechanical properties    of some polyethers. Polymer 1966, 7 (7), 331-365; (b) Jones, D. S.;    Bruschi, M. L.; de Freitas, O.; Gremião, M. P. D.; Lara, E. H. G.;    Andrews, G. P., Rheological, mechanical and mucoadhesive properties    of thermoresponsive, bioadhesive binary mixtures composed of    poloxamer 407 and carbopol 974P designed as platforms for    implantable drug delivery systems for use in the oral cavity.    International Journal of Pharmaceutics 2009, 372 (1-2), 49-58.-   43. Wan, A. C. A.; Mao, H.-Q.; Wang, S.; Phua, S. H.; Lee, G. P.;    Pan, J.; Lu, S.; Wang, J.; Leong, K. W., Poly(phosphoester) ionomers    as tissue-engineering scaffolds. Journal of Biomedical Materials    Research Part B: Applied Biomaterials 2004, 70B (1), 91-102.-   44. Iwasaki, Y.; Wachiralarpphaithoon, C.; Akiyoshi, K., Novel    Thermoresponsive Polymers Having Biodegradable Phosphoester    Backbones. Macromolecules 2007, 40 (23), 8136-8138.-   45. (a) Wen, J.; Mao, H.-Q.; Li, W.; Lin, K. Y.; Leong, K. W.,    Biodegradable polyphosphoester micelles for gene delivery. Journal    of Pharmaceutical Sciences 2004, 93 (8), 2142-2157; (b) Li, Q.;    Wang, J.; Shahani, S.; Sun, D. D. N.; Sharma, B.; Elisseeff, J. H.;    Leong, K. W., Biodegradable and photocrosslinkable polyphosphoester    hydrogel. Biomaterials 2006, 27 (7), 1027-1034; (c) Zhao, Z.; Wang,    J.; Mao, H.-Q.; Leong, K. W., Polyphosphoesters in drug and gene    delivery. Advanced Drug Delivery Reviews 2003, 55 (4), 483-499.-   46. McCormick, C. L.; Sumerlin, B. S.; Lokitz, B. S.; Stempka, J.    E., RAFT-synthesized diblock and triblock copolymers:    thermally-induced supramolecular assembly in aqueous media. Soft    Matter 2008, 4 (9), 1760-1773.-   47. Bromberg, L., Properties of Aqueous Solutions and Gels of    Poly(ethylene oxide)-b-poly(propylene oxide)-b-poly(ethylene    oxide)-g-poly(acrylic acid). The Journal of Physical Chemistry B    1998, 102 (52), 10736-10744.-   48. (a) Dumortier, G.; Grossiord, J. L.; Agnely, F.; Chaumeil, J.    C., A review of poloxamer 407 pharmaceutical and pharmacological    characteristics. Pharm Res 2006, 23 (12), 2709-28; (b)    Barreiro-Iglesias, R.; Bromberg, L.; Temchenko, M.; Hatton, T. A.;    Alvarez-Lorenzo, C.; Concheiro, A., Pluronic-g-poly(acrylic acid)    copolymers as novel excipients for site specific, sustained release    tablets. European Journal of Pharmaceutical Sciences 2005, 26 (5),    374-385; (c) Cole, M. L.; Whateley, T. L., Interaction of Nonionic    Block Copolymeric (Poloxamer) Surfactants with Poly (Acrylic Acid),    Studied by Photon Correlation Spectroscopy. Journal of Colloid and    Interface Science 1996, 180 (2), 421-427.-   49. Kohane, D. S.; Plesnila, N.; Thomas, S. S.; Le, D.; Langer, R.;    Moskowitz, M. A., Lipid-sugar particles for intracranial drug    delivery: safety and biocompatibility. Brain Res 2002, 946 (2),    206-13.-   50. Gabriel, D.; Monteiro, I. P.; Huang, D.; Langer, R.; Kohane, D.    S., A photo-triggered layered surface coating producing reactive    oxygen species. Biomaterials 2013, 34, 9763-9769.-   51. Sabharwal, V.; Ram, S.; Figueira, M.; Park, I. H.; Pelton, S.    I., Role of complement in host defense against pneumococcal otitis    media. Infect Immun 2009, 77 (3), 1121-7.-   52. Novotny, L. A.; Clements, J. D.; Bakaletz, L. O., Kinetic    analysis and evaluation of the mechanisms involved in the resolution    of experimental nontypeable Haemophilus influenzae-induced otitis    media after transcutaneous immunization. Vaccine 2013, 31 (34),    3417-26.

EQUIVALENTS AND SCOPE

In the claims articles such as “a,” “an,” and “the” may mean one or morethan one unless indicated to the contrary or otherwise evident from thecontext. Claims or descriptions that include “or” between one or moremembers of a group are considered satisfied if one, more than one, orall of the group members are present in, employed in, or otherwiserelevant to a given product or process unless indicated to the contraryor otherwise evident from the context. The invention includesembodiments in which exactly one member of the group is present in,employed in, or otherwise relevant to a given product or process. Theinvention includes embodiments in which more than one, or all of thegroup members are present in, employed in, or otherwise relevant to agiven product or process.

Furthermore, the invention encompasses all variations, combinations, andpermutations in which one or more limitations, elements, clauses, anddescriptive terms from one or more of the listed claims is introducedinto another claim. For example, any claim that is dependent on anotherclaim can be modified to include one or more limitations found in anyother claim that is dependent on the same base claim. Where elements arepresented as lists, e.g., in Markush group format, each subgroup of theelements is also disclosed, and any element(s) can be removed from thegroup. It should it be understood that, in general, where the invention,or aspects of the invention, is/are referred to as comprising particularelements and/or features, certain embodiments of the invention oraspects of the invention consist, or consist essentially of, suchelements and/or features. For purposes of simplicity, those embodimentshave not been specifically set forth in haec verba herein. It is alsonoted that the terms “comprising” and “containing” are intended to beopen and permits the inclusion of additional elements or steps. Whereranges are given, endpoints are included. Furthermore, unless otherwiseindicated or otherwise evident from the context and understanding of oneof ordinary skill in the art, values that are expressed as ranges canassume any specific value or sub-range within the stated ranges indifferent embodiments of the invention, to the tenth of the unit of thelower limit of the range, unless the context clearly dictates otherwise.

This application refers to various issued patents, published patentapplications, journal articles, and other publications, all of which areincorporated herein by reference. If there is a conflict between any ofthe incorporated references and the instant specification, thespecification shall control. In addition, any particular embodiment ofthe present invention that falls within the prior art may be explicitlyexcluded from any one or more of the claims. Because such embodimentsare deemed to be known to one of ordinary skill in the art, they may beexcluded even if the exclusion is not set forth explicitly herein. Anyparticular embodiment of the invention can be excluded from any claim,for any reason, whether or not related to the existence of prior art.

Those skilled in the art will recognize or be able to ascertain using nomore than routine experimentation many equivalents to the specificembodiments described herein. The scope of the present embodimentsdescribed herein is not intended to be limited to the above Description,but rather is as set forth in the appended claims. Those of ordinaryskill in the art will appreciate that various changes and modificationsto this description may be made without departing from the spirit orscope of the present invention, as defined in the following claims.

1. A composition comprising: (a) a therapeutic agent or a combination oftherapeutic agents; (b) a permeation enhancer or a combination ofpermeation enhancers, wherein the permeation enhancer or combination ofpermeation enhancers increases the flux of the therapeutic agent orcombination of therapeutic agents across a barrier; and (c) a matrixforming agent or a combination of matrix forming agents, wherein thematrix forming agent or combination of matrix forming agents comprises apolymer; wherein: the composition forms a gel at temperatures above aphase transition temperature; and the phase transition temperature isless than about 37° C.; and at least one of conditions (i), (ii), and(iii) are met: (i) the phase transition temperature of the compositionis less than the phase transition temperature of a reference compositionplus about 5° C.; (ii) the storage modulus of the composition is greaterthan about 15% of the storage modulus of the reference composition orgreater than about 500 Pa, whichever is smaller, at a temperature ofabout 37° C.; and (iii) the loss modulus of the composition is betweenabout 15% and about 150% of the loss modulus of the referencecomposition at a temperature of about 37° C.; wherein the referencecomposition is the composition in the absence of the permeation enhanceror combination of permeation enhancers. 2-15. (canceled)
 16. Thecomposition of claim 1, wherein the polymer is a block copolymercomprising at least one hydrophobic block. 17-25. (canceled)
 26. Thecomposition of claim 1, wherein the polymer comprises phosphoestermonomers of Formula (M):

wherein for each monomer Y is independently —R¹ or -L²R², wherein: eachoccurrence of R¹ is independently optionally substituted alkyl,optionally substituted alkenyl, optionally substituted alkynyl,optionally substituted carbocyclyl, optionally substituted heterocyclyl,optionally substituted aryl, optionally substituted heteroaryl,optionally substituted acyl, or an oxygen protecting group; eachoccurrence of L² is independently optionally substituted alkylene,optionally substituted alkenylene, optionally substituted alkynylene,optionally substituted heteroalkylene, optionally substitutedheteroalkenylene, or optionally substituted heteroalkynylene; eachoccurrence of R² is independently optionally substituted acyl,optionally substituted carbocyclyl, optionally substituted heterocyclyl,optionally substituted aryl, optionally substituted heteroaryl, —OR^(b),—N(R^(b))₂; and each occurrence of R^(b) is independently optionallysubstituted alkyl, optionally substituted alkenyl, optionallysubstituted alkynyl, optionally substituted carbocyclyl, optionallysubstituted heterocyclyl, optionally substituted aryl, optionallysubstituted heteroaryl, optionally substituted acyl, an oxygenprotecting group, or a nitrogen protecting group, or two R^(b) takentogether with the nitrogen to which they are attached form an optionallysubstituted heterocyclic or optionally substituted heteroaryl ring.27-32. (canceled)
 33. The composition of claim 1, wherein the polymer isof formula:

wherein: each occurrence of Y is independently —R¹ or -L²R²; eachoccurrence of R¹ is independently hydrogen, optionally substitutedalkyl, optionally substituted alkenyl, optionally substituted alkynyl;each occurrence of L² is independently a bond, optionally substitutedalkylene, optionally substituted alkenylene, optionally substitutedalkynylene, optionally substituted heteroalkylene, optionallysubstituted heteroalkenylene, or optionally substitutedheteroalkynylene; each occurrence of R² is independently optionallysubstituted acyl, optionally substituted carbocyclyl, optionallysubstituted heterocyclyl, optionally substituted aryl, optionallysubstituted heteroaryl, —OR^(b), —N(R^(b))₂, or an oxygen protectinggroup; each occurrence of R³ is independently optionally substitutedalkyl, optionally substituted alkenyl, optionally substituted alkynyl,optionally substituted aryl, optionally substituted heteryaryl,optionally substituted acyl, —OR^(b), or —N(R^(b))₂; each occurrence ofR^(b) is independently optionally substituted alkyl, optionallysubstituted alkenyl, optionally substituted alkynyl, optionallysubstituted carbocyclyl, optionally substituted heterocyclyl, optionallysubstituted aryl, optionally substituted heteroaryl, optionallysubstituted acyl, an oxygen protecting group, or a nitrogen protectinggroup, or two R^(b) taken together with the nitrogen to which they areattached form an optionally substituted heterocyclic or optionallysubstituted heteroaryl ring; each of G¹ and G² is independentlyhydrogen, optionally substituted alkyl, optionally substituted acyl,optionally substituted phosphate, or an oxygen protecting group; andeach of p, q, r, s, and t is independently an integer between 0 and 200,wherein the sum of p and t is at least 1, and the sum of q, r, and s isat least
 1. 34. The composition of claim 33, wherein the polymer is ofthe formula:


35. The composition of claim 1, wherein the polymer is of the formula:


36. The composition of claim 33, wherein each R¹ is unsubstituted C₁₋₂₀alkyl.
 37. The composition of claim 33, wherein each L² is a bond orunsubstituted C₁₋₆ alkylene. 38-41. (canceled)
 42. The composition ofclaim 1, wherein the permeation enhancer is a surfactant, terpene, aminoamide, amino ester, azide-containing compound, alcohol, pyrrolidone,sulfoxide, fatty acid, or anesthetic agent. 43-54. (canceled)
 55. Thecomposition of claim 1, wherein the therapeutic agent is an antibioticagent, anesthetic agent, anti-inflammatory agent, analgesic agent,anti-fibrotic agent, anti-sclerotic agent, anticoagulant agent, ordiagnostic agent. 56-64. (canceled)
 65. A matrix forming agent or acombination of matrix forming agents, wherein the matrix forming agentor combination of matrix forming agents comprises a polymer of Formula(I′):

wherein: each occurrence of Y is independently —R¹ or -L²R²; eachoccurrence of R¹ is independently hydrogen, optionally substitutedalkyl, optionally substituted alkenyl, optionally substituted alkynyl,optionally substituted aryl, or optionally substituted heteroaryl; eachoccurrence of L² is independently a bond, optionally substitutedalkylene, optionally substituted alkenylene, optionally substitutedalkynylene, optionally substituted heteroalkylene, optionallysubstituted heteroalkenylene, or optionally substitutedheteroalkynylene; each occurrence of R² is independently optionallysubstituted acyl, optionally substituted carbocyclyl, optionallysubstituted heterocyclyl, optionally substituted aryl, optionallysubstituted heteroaryl, —OR^(b), —N(R^(b))₂, or an oxygen protectinggroup; each occurrence of R^(3A) is independently hydrogen, optionallysubstituted alkyl, optionally substituted alkenyl, optionallysubstituted alkynyl, optionally substituted aryl, optionally substitutedheteryaryl, optionally substituted acyl, —OR^(b), or —N(R^(b))₂; eachoccurrence of R^(b) is independently optionally substituted alkyl,optionally substituted alkenyl, optionally substituted alkynyl,optionally substituted carbocyclyl, optionally substituted heterocyclyl,optionally substituted aryl, optionally substituted heteroaryl,optionally substituted acyl, an oxygen protecting group, or a nitrogenprotecting group, or two R^(b) taken together with the nitrogen to whichthey are attached form an optionally substituted heterocyclic oroptionally substituted heteroaryl ring; each of G^(1A) and G^(2A) isindependently hydrogen, halogen, optionally substituted amine,optionally substituted alkyl, optionally substituted aryl, or optionallysubstituted heteroaryl, optionally substituted acyl, optionallysubstituted phosphate, or an oxygen protecting group; and each of p, q,r, s, and t is independently an integer between 0 and 200, inclusive,wherein the sum of p and t is at least 1, and the sum of q, r, and s isat least
 1. 66. The polymer of claim 65, wherein the matrix formingagent or combination of matrix forming agents comprises a polymer ofFormula:

wherein: each occurrence of Z is independently —R⁴; each occurrence ofR⁴ is independently optionally substituted alkyl; each of G^(1A) andG^(2A) is independently hydrogen, optionally substituted alkyl,optionally substituted aryl, or optionally substituted heteroaryl,optionally substituted acyl, optionally substituted phosphate, or anoxygen protecting group; and each of p, q, r, s, and t is independentlyan integer between 0 and 200, wherein the sum of p and t is at least 1,and the sum of q, r, and s is at least 1; the composition forms a gel attemperatures above a phase transition temperature; and the phasetransition temperature is less than about 37° C.; and at least one ofconditions (i), (ii), and (iii) are met: (i) the phase transitiontemperature of the composition is less than the phase transitiontemperature of a reference composition plus about 5° C.; (ii) thestorage modulus of the composition is greater than about 15% of thestorage modulus of the reference composition at a temperature of about37° C.; and (iii) the loss modulus of the composition is between about80% and about 120% of the loss modulus of the reference composition at atemperature of about 37° C.; wherein the reference composition is thecomposition in the absence of the permeation enhancer or combination ofpermeation enhancers. 67-109. (canceled)
 110. A method of treating aninfectious disease, comprising administering a composition of claim 1,to a subject in need thereof.
 111. A method of treating an ear disease,comprising administering a composition of claim 1, to a subject in needthereof.
 112. The method of claim 110, wherein the infectious disease isa bacterial infection.
 113. (canceled)
 114. The method of claim 110,wherein the infectious disease is otitis media.
 115. A method oferadicating a biofilm, comprising administering a composition of claim1, to a subject in need thereof.
 116. A method of delivering acomposition of claim 1, the method comprising administering thecomposition to an ear canal of a subject, wherein the compositioncontacts the surface of a tympanic membrane.
 117. A method of deliveringa composition of claim 1, the method comprising administering thecomposition to an ear canal of a subject. 118-128. (canceled)
 129. A kitfor treating an ear disease comprising a container, a composition ofclaim 1, and instructions for administering the composition to a subjectin need thereof. 130-131. (canceled)